# Patent

## Title
Nanoparticles for immunotherapy

## Abstract

Nanoparticles that activate complement in the absence of biological molecules are described. The nanoparticles are shown to specifically target antigen presenting cells in specifically in lymph nodes, without the use of a biological molecule for targeting. These particles are useful vehicles for delivering immunotherapeutics. Surface chemistries and chemical formulations for the nanoparticles are described.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is related to U.S. application Ser. No. 11/707,627, which is hereby incorporated by reference herein.
TECHNICAL FIELD
The technical field relates, in some aspects, to nanoparticles with surface chemistries for activating the immune system.
BACKGROUND
Many medical benefits could be realized if the immune system could be trained to respond to antigens in a desired manner such as by developing tolerance to the antigen or learning to reject it. Diverse approaches have been attempted to meet this challenge, including systemic drug treatments, injection of antigens, and antibody therapies.
SUMMARY OF THE INVENTION
One new approach, among others disclosed herein, specifically targets therapeutic substances to antigen-presenting cells (APCs) at a specific location in the body. APCs are typically dendritic cells and macrophages and in some cases B cells. Herein, APC is a term used only to describe dendritic cells and macrophages, and excludes B cells. Even though APCs are spread throughout the body, this approach targets the agent to the APCs at a particular location: the lymph node. APCs behave differently at the lymph node as opposed to other parts of the body, so that intake of the agent at this location is advantageous. Further, the vehicle persists over hours or days so that it can accomplish its effects and is also biodegradable. Not only are APCs targeted specifically at the lymph node, but the delivery vehicle for the therapeutic agent can be used to activate the APCs in a specific manner: by activating the complement system. Activating the complement system invokes known pathways of response so that appropriate immunotherapeutic agents can be chosen. Moreover, the complement system can be activated by synthetic materials in the delivery vehicle without involving biological agents. The result of all of these specially-targeted features is a vehicle that generically delivers a therapeutic agent to APCs at a time and place wherein the APCs are activated to achieve a desired immunotherapy. The vehicle itself does not require biological molecules or polypeptides so that it is ready to receive any agent without conflict, cross-reaction, or unwanted antagonism of the immune system.
This approach includes, in some embodiments, particles that have suitable physical properties and that are sized to flow through the interstitial spaces to penetrate the lymphatic system. Particles that are too large will not effectively migrate to the lymphatic system. Such particles may be made with biodegradable synthetic polymers and polymers that activate complement. Such particles may be made by crosslinking various polymers together and disposing certain complement-activating functional groups at a location on the particles that is available for complement activation. All of these features are described in detail, below.
In some embodiments, a composition is a nanoparticle composition comprising: an isolated collection of synthetic biodegradable particles associated with an immunotherapeutic agent and comprising a first polymer that activates complement and a second covalently crosslinked polymer, wherein the collection has a mean diameter from about 10 nm to about 100 nm, or less than about 70 nm, the first polymer is free of naturally-occurring biomolecules that activate complement, and the first polymer is strongly bound to the second polymer.
Some embodiments relate to a method of making an immunotherapeutic composition of nanoparticles comprising emulsion polymerization of a first polymer with a second polymer that is the emulsifier used during the polymerization to make a collection of biodegradable particles with a mean diameter of between about 20 nm and about 100 nm, or less than about 70 nm, choosing the second polymer to comprise hydroxyl and/or thiol functional groups that activate complement, and associating an immunotherapeutic with the particles.
Some embodiments relate to a method of delivering an immunotherapeutic agent, the method comprising introducing into a patient a collection of synthetic biodegradable particles that are specifically targeted to antigen presenting cells in lymph nodes, wherein the particles comprise a first polymer that activates complement, the collection has a mean diameter from about 10 nm to about 100 nm, or less than about 70 nm, the first polymer is free of naturally-occurring biomolecules that activate complement, and the particles comprise a second covalently crosslinked polymer that is strongly bound to the first polymer.
Some embodiments relate to a nanoparticle composition comprising an isolated collection of synthetic particles that comprise a synthetic polymer that activates complement, wherein the collection has a mean particle diameter of, e.g., about 10 nm to about 100 nm, or less than about 70 nm. The particles may further be associated with an antigen. The synthetic polymer in some embodiments is free of sequences of amino acids or sequences of saccharides that activate complement, or altogether free of amino acids and/or nucleic acids and/or saccharides. The synthetic polymer may, e.g., comprise a hydrophobic portion that is adsorbed to a hydrophobic portion of a second biodegradable polymer that forms a core of the nanoparticle to thereby bind the synthetic polymer to the core.
Some embodiments relate to nanoparticle composition comprising: an isolated collection of synthetic particles, wherein the collection has a mean diameter of about, e.g., 10 nm to about 100 nm, or less than about 70 nm, wherein the particles comprise an immunosuppressant drug or other agent, and wherein the particles are further associated with an antigen. In some versions, the particles comprise an amphiphilic block copolymer of at least one hydrophobic block and at least one hydrophilic block, wherein the block copolymer self-assembles in aqueous solutions to form the particles.
Some embodiments relate to a medical composition for introduction into a mammal comprising a collection of synthetic nanoparticles that have a mean particle diameter of less than about 70 nm that comprise an antigen and a synthetic polymer with functional groups disposed on an exterior of the nanoparticle that activate complement in a mammal. The functional groups may comprise, e.g., nucleophilic, e.g., hydroxyls and/or thiols. The functional groups may be chosen to induce a Th-1 response in a mammal as applied in the context of a nanoparticle system.
The nanoparticles may be micelles. Some micellar embodiments are made with a block copolymer comprising a hydrophilic polymer and a hydrophobic polymer. An example of the hydrophilic polymer is one that is water soluble and comprises polyethylene oxide. An exemplary hydrophobic polymer is a polymer that is not water soluble and comprises poly(propylene sulfide). A therapeutic agent may be encapsulated with the micelle. The compositions, generally, may be free of an adjuvant other than the synthetic polymer.
Nanoparticles may be associated with one or more antigens or a danger signal of which a variety are detailed herein. Some such associates involve a covalent, ionic, and/or a disulfide bond. The danger signal may be a bioactive molecule other than the functional groups or the polymers used to form the nanoparticle; in other words, formulations for making nanoparticles may be further modified with another molecule that is a danger signal, be it one found in nature or not found in nature. Some danger signals are hydrophobic and may be associated with hydrophobic portions of the nanoparticles. A method of association is a charge-charge interaction, with the moiety to be associated to the particle having one charge and the nanoparticle, before the association, having the opposite charge, e.g., a positive charge on the nanoparticle and negative charges on the moiety to be associated. For instance, a peptide may be expressed with a plurality of positive or negatively charged moieties, e.g., aspartate and/or glutamate (with the charge state referring to typical in vivo pH conditions). Examples of antigens include a tumor antigen or an antigen of an infectious disease, e.g., Caspase-8, MAGE-1, Tyrosinase, HER-2/neu, or MUC-1. Examples of a danger signal are inflammatory cytokines or ligands for Toll-like receptors. Nanoparticles may be free of targeting ligands that specifically bind to a cell
In some embodiments, the nanoparticle is made using a synthetic polymer that comprises an amphiphilic block copolymer of at least one hydrophobic block and at least one hydrophilic block, wherein the block copolymer self-assembles in aqueous solutions to form the particles, wherein functional groups comprise hydroxyls and/or thiols. Or the nanoparticles can comprise a core and a hydrophilic corona, wherein the core is comprised of poly(propylene sulfide) and the hydrophilic corona is comprised of the synthetic polymer which comprises polyethylene glycol, and wherein the functional groups comprise nucleophiles.
One embodiment is an immunostimulatory composition for introduction into a patient that comprises a collection of synthetic nanoparticles that have a mean particle diameter of less than about 70 nm and comprise an antigen, with the composition inducing an immune response against the antigen in the patent without inducing a TNF-alpha response and without inducing an IL-6 response. Such nanoparticles may have features as described herein, e.g., the nanoparticles are free of targeting ligands that specifically bind to a cell, and/or the antigen is an antigen of a tumor or an infectious disease.
The nanoparticles may further comprise an immunosuppressant. This feature is advantageous for inducing tolerization, with vaccination with an antigen in the complete absence of DC activation inducing tolerance. Thus the nanoparticle delivers antigen but also highly specific and thus highly effective, immunosuppression of the affected dendritic cells
Another embodiment relates to a medical composition for introduction into a mammal comprising a collection of synthetic nanoparticles that have a mean particle diameter of less than about 70 nm that comprise a nucleic acid encoding an antigen and a synthetic polymer with functional groups disposed on an exterior of the nanoparticle that activate complement in a mammal.
An embodiment is a composition or a method of transfecting a dendritic cell in a lymph node of a patient comprising administering to a patient, in a location external to a lymph node, a collection of synthetic nanoparticles that comprise a nucleic acid encoding an antigen, with the nanoparticles having a mean particle diameter of less than about 70 nm and a surface chemistry for traveling to the lymph node of the patient for uptake by dendritic cells to transfect the dendritic cells with the nucleic acid, with the surface chemistry comprising a plurality of nucleophilic groups associated with a synthetic hydrophilic polymer that activates complement. The efficiency of transfection may be e.g., at least about 30% of the dendritic cells in the lymph node are transfected with the nucleic acid. Various of the nanoparticle embodiments are available for this case, e.g., wherein the nanoparticles comprise a core of poly(propylene sulfide), the synthetic hydrophilic polymer comprises polyethylene glycol, and the functional groups comprise hydroxyls and/or thiols, or wherein the nanoparticles comprise a core and a hydrophilic corona that comprises the hydrophilic polymer and the nucleophilic groups, wherein the core is comprised of poly(propylene sulfide), or wherein the nanoparticles comprise micelles.
Another embodiment is a method, or a composition for introduction into a patient comprising a branched polymer conjugated to an antigen, wherein the branched polymer comprises at least for termini that are nucleophilic groups and activates complement after introduction into a patient, and wherein the composition stimulates an immune system of the patient to respond to the antigen. For instance, the branched polymer may comprise polyethylene glycol and the nucleophilic groups may be hydroxyls or thiols.
An embodiment is a composition or a method of simulating a dendritic cell in a lymph node of a patient comprising administering to a patient, in a location external to a lymph node, a branched polymer that activates complement, wherein the polymer is conjugated to an antigen and comprises at least four termini that are nucleophilic groups, with the polymer traveling to the lymph node of the patient for uptake by dendritic cells to present the antigen.
An embodiment is a composition or a method of stimulating a dendritic cell in a lymph node of a patient comprising administering to a patient, in a location external to a lymph node, a collection of synthetic branched polymers that activate complement and comprise an antigen, with the polymers further comprising at least four termini that comprise a nucleophilic group for uptake by dendritic cells at a lymph node and release of the antigen to the dendritic cell, with the surface chemistry comprising a plurality of nucleophilic groups associated with a synthetic hydrophilic polymer.
An embodiment is a composition or a method of delivering a medical vaccine comprising providing a collection of nanoparticles. For instance, a method of transfecting a dendritic cell in a lymph node of a patient comprising administering to an external mucosal surface of a patient, under mild pressure, a collection of synthetic nanoparticles that comprise an antigen, with the nanoparticles having a mean particle diameter of less than about 70 nm and a surface chemistry for traveling to the lymph node of the patient for uptake by dendritic cells to transfect the dendritic cells with the nucleic acid, with the surface chemistry comprising a plurality of nucleophilic groups associated with a synthetic hydrophilic polymer that activates complement. An example of the mucosal surface is nasal mucosa.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a photomicrographic montage showing a comparison of nanoparticle uptake into the initial lymphatics. It depicts fluorescence microlymphangiography of the lymphatic capillary network in mouse tail skin after 90 min infusion with fluorescence-loaded PPS nanoparticles of (A) 20, (B) 45 and (C) 100 nm diameter. Images were taken at constant exposure. The hexagonal lymphatic network was clearly visible with the 20 nm particles. Bar=100 μm.
FIG. 2 is a photomicrographic montage showing lymph node retention of nanoparticles. Shown are sections from draining lymph nodes following injections into the mouse tail with 20, 45, and 100 nm PPS nanoparticles. Lymph nodes were removed at 24, 72, 96, and 120 h post-injection. Nanoparticles were strongly present at all time points for 20 and 45 nm nanoparticles, but 100 nm particles were not seen in the lymph nodes. Bar=200 μm.
FIG. 3 is a photomicrographic montage showing localization of nanoparticles within the lymph node. Shown are serial lymph node sections at 96 h following injection of 20 nm PPS nanoparticles. Immune cells were identified as indicated with antibodies against (A) CD3e (T cells), (B) CD45R (B cells), and (C) CD68 (macrophages (Macs) and dendritic cells (DCs)). Nanoparticles are distinctly absent in T cell and B cell zones, but strongly co-localized with macrophages and DCs. Bar=100 μm. (D) The endothelial marker CD31 demonstrates nanoparticle (green) distribution relative to lymph node sinus architecture. Bar=100 μm.
FIG. 4 is a photomicrographic montage showing internalization of nanoparticles by macrophages and dendritic cells (DCs). Shown are confocal images of lymph node sections at 96 h following injection of 20 nm PPS nanoparticles. (A) Staining for CD68, expressed by both macrophages and DCs, reveals internalization of nanoparticles by these cells. (B) Staining for Dec-205, which is found exclusively on DCs, demonstrates that DCs also internalize the nanoparticles. Bar=20 μm.
FIG. 5 has bar graphs that show quantification of cell uptake of nanoparticles (NPs). Flow cytometry analysis was used to determine the fraction of lymph node APCs (MHCII+) and DCs (CD11c+) that internalized NPs (FITC+). (A) At 24 h, following injections, more than 38% of APCs and 50% of DCs in lymph nodes had internalized 20 nm nanoparticles. There was reduced uptake in both cell populations with 45 nm nanoparticles, and only 10% of all APCs took up 100 nm nanoparticles. (B) After in vitro pulsing of APCs and DCs with nanoparticles for 24 h, nearly all APCs and DCs had internalized nanoparticles of all 3 sizes. Thus since all 3 nanoparticle sizes are equally taken up in vitro, the differences seen following in vivo injection are most likely due to differences in nanoparticle uptake into lymphatics following injection. These results also indicate that nanoparticle uptake occurs in the lymph nodes rather than by cells in peripheral sites, which then migrate to lymph nodes.
FIG. 6 is a (A) a photomicrographic montage and (B) bar graph that both show increase of presence of macrophages and dendritic cells (DCs) with time. Shown are lymph node sections stained for macrophages (Macs) and DCs (CD68+ cells) and nuclei at 24 and 96 h post-injection of 20 nm PPS nanoparticles (NPs). Mac and DC co-localization increased with time. Bar=100 μm. (B) Flow cytometry analysis was used to determine the fraction of lymph node (LN) cells with nanoparticles (NPs+) that were APCs (MHCII+) and DCs (CD11c+) at 24 and 96 h post-injection of 20 nm nanoparticles. There is a significant increase in the fraction of cells with nanoparticles that are APCs and DCs at 96 h vs. 24 h. Also, it appears that nearly all APCs with nanoparticles at 24 h are DCs.
FIG. 7 depicts graphs that show an increase in expression of DC maturation markers, CD86 and CD80 following nanoparticle (NP) internalization. (A) A typical histogram of CD86 expression of DCs (CD11c+) at 24 h post-injection with PBS or 20 nm particles. A clear shift in CD86 expression is observed for DCs with nanoparticles (CD11c+FITC+). (B) The fraction of cells positively expressing CD86 and CD80 is determined to be significantly greater following nanoparticle internalization. Additionally it is shown that CD86 and CD80 expression remains at higher levels at 96 h post-injection.
FIG. 8 depicts data showing that PLURONIC F-127 is modified so that the terminal OH groups are converted to OCH3 groups (A). (B) Incubation of PLURONIC-stabilized (and thus hydroxylated) complement-activating nanoparticles (OH—NPs) with serum causes greater complement activation than nanoparticles stabilized with methoxy-terminated PLURONIC (CH3—NPs) measured through fold increase of C3a in serum+PBS.
FIG. 9 depicts graphs showing that nanoparticle surface chemistry dictates DC maturation response. 25 nm PLURONIC-stabilized (and thus hydroxylated) complement activating nanoparticles (OH—NPs) mature DCs to a much greater extent than 25 nm nanoparticles stabilized with methoxy-terminated PLURONIC (CH3—NPs) and 20 nm carboxylated polystyrene nanospheres (COOH—NSs).
FIG. 10 depicts graphs showing that nanoparticle size dictates DC maturation response. 25 nm PLURONIC-stabilized (and thus hydroxylated) complement activating nanoparticles (OH—NPs) induce DC maturation, whereas 100 nm PLURONIC-stabilized (and thus hydroxylated) complement activating nanoparticles do not.
FIG. 11 shows a chemical scheme to modify nanoparticles and a photomicrograph of the same in lymph nodes, wherein at (A) PLURONIC is functionalized with vinylsulfone (PL-VS). Vinylsulfone can then be attached to the free cysteines on Ovalbumin (OVA). PL-VS-OVA is then blended with PLURONIC and 25 nm nanoparticles are synthesized as per usual. (B) 25 nm OVA-conjugated PLURONIC-stabilized nanoparticles deliver OVA to lymph nodes.
FIG. 12 depicts graphs showing that 25 nm OVA-conjugated PLURONIC-stabilized (and thus hydroxylated) complement activating PPS nanoparticles (OH-OVA-NPs) induce DC maturation to the similar levels as OVA with LPS following injections at 24 hr.
FIG. 13 depicts graphs showing that 25 nm OVA-conjugated PLURONIC-stabilized (and thus hydroxylated) complement activating nanoparticles (OH-OVA-NPs) cause CD4 T cell proliferation at same levels as OVA with LPS after adoptive transfer of T cells from OT-II mice.
FIG. 14 depicts graphs showing that 25 nm OVA-conjugated PLURONIC-stabilized (and thus hydroxylated) complement activating nanoparticles (OH-OVA-NPs), cause CD8 T cell memory, measured through IFN-γ spots/lymph node cells. *, P<0.05.
FIG. 15 depicts graphs showing OVA Ab titers at 21 days. 25 nm OVA-conjugated PLURONIC-stabilized (and thus hydroxylated) nanoparticles (OH-OVA-NPs) cause OVA Ab titers at levels similar to OVA with LPS. 25 nm OVA-conjugated methoxy-terminated PLURONIC stabilized PPS nanoparticles and 100 nm OVA-conjugated PLURONIC-stabilized (and thus hydroxylated) nanoparticles (OH-OVA-100) cause lower Ab titers. 25 nm OVA-conjugated PLURONIC-stabilized (and thus hydroxylated) (OH-OVA-25) cause lower Ab titers in C3−/− mice.
FIG. 16 depicts graphs showing that complement-activating nanoparticles induce cellular immunity with a Th1 biased response. Panel a is a bar graph that displays production of interleukin 12p40 (IL-12p40) by bone marrow derived dendritic cells (BMDCs) following a 24 h incubation with nanoparticles and controls. IL-12p40 was measured by ELISA on cell media, fold increase is of serum. Panel b is a bar graph that displays MHC I expression (measured by flow cytometry) following 24 h incubation of BMDCs with nanoparticles and controls. Panel c is a bar graph showing results of a re-stimulation assay used to determine CD8 T cell memory. Mice received various injections of antigen and nanoparticle formulations and draining lymph nodes were isolated at 21 d post-injection. The graph shows interferon-gamma (IFN-g) producing CD8 T cells after exposure to MHC-I OVA antigenic peptide and demonstrates memory after treatment with OH-OVA-25-NPs but not CH3O-OVA-25-NPs. Results are mean values from three mice in each group, error bars correspond to SEM. *, P<0.05.
FIG. 17 shows results for complement-activating nanoparticles do not induce pro-inflammatory cytokines. Panels a,b are bar graphs that display production of tumor necrosis factor-alpha (TNF) and interleukin-6 (IL-6), respectively by bone marrow derived dendritic cells following a 24 h incubation with nanoparticles and controls. Cytokines were measured by ELISA on cell media. N.D. (not detectable).
FIG. 18 shows molecular measurements of hydroxylated nanoparticle IL-12p40 secretion in vitro and in anti-OVA titers in vivo. Panel a is a bar graph showing high IL-12p40 secretion of serum-exposed hydroxylated nanoparticles in mice with TLR4 versus low secretion mice without TLR4. Panel b is a logarithmic scatterplot of antibodies against ovalbumin (OVA) induced by introduction of hydroxylated nanoparticles into mice with TLR4 (wildtype) or without TLR4 (TLR4−/−). Mice without TLR4 had much lower levels of antibodies against OVA, indicating that TLR4 is an important mediator for mechanisms of antibody induction by hydroxylated nanoparticles.
FIG. 19 shows that hydroxyl and methoxy nanoparticles with thiol surfaces (OH—SH— and CH3-SH—NPs, respectively) activate complement significantly more than nanoparticles without thiols. Nanoparticle-induced complement activation, as measured through C3a presence in human serum following incubation with nanoparticles, was demonstrated to be much higher with OH—SH—NPs and CH3-SH—NPs compared to nanoparticles without thiols (OH— and CH3O—NPs). Results are normalized to control of serum incubation with PBS. Values are means of three independent experiments; error bars correspond to standard error of mean, SEM.
FIG. 20 shows that nanoparticles with hydroxy and thiol surfaces (OH—SH—NP) activate dendritic cells. Panel a, Flow cytometry histogram shows CD80 expression, a marker of maturation on bone marrow derived dendritic cells (BMDCs) following exposure to nanoparticles and controls. OH—SH—NPs with serum (complement) induce dendritic cell maturation while hydroxy nanoparticles without thiols (OH—NP) and serum do not. Panel b, Bar graph displays production of interleukin 12p40 (IL-12p40) by BMDCs following a 24 h incubation with nanoparticles and controls. IL-12p40 was measured by ELISA on cell media, fold increase is of serum. OH—SH—NP with serum cause high levels of IL-12p40 while OH—NPs with serum do not.
FIG. 21 is a flow cytometry histogram showing that PEG-PPS nanoparticles loaded with imiquimod demonstrated strong maturation of bone marrow derived dendritic cells (BMDCs). Flow cytometry histogram of maturation marker CD86 shows that PEG-PPS micelles loaded with imiquimod matures BMDCs, approaching that of potent activator LPS.
FIG. 22 depicts a synthetic route for production of PEG-PPS block copolymer amphiphile, from which polymer micelles can be formed. A route by which to obtain a —OH group on the terminus of the PEG chain is shown. Without end-capping, a terminal —SH is obtained, and with end-capping the terminal —SH group can be blocked.
FIG. 23 depicts a synthetic route for production of PEG-PPS block copolymer amphiphile, from which polymer micelles can be formed. A route by which to obtain a polymer with a —CH3 group on the terminus of the PEG chain, i.e. lacking a —OH group on the terminus of the PEG chain, is shown. As in FIG. 22, the terminal —SH chain can be preserved or blocked.
FIG. 24A depicts a synthetic route for production of an amine-containing PEG-PPS block copolymer amphiphile, from which polymer micelles can be formed, to which antigen can be coupled. The case of a polymer with a —CH3 group on the terminus of the PEG chain is shown.
FIG. 24B depicts a synthetic route for production in the case of a polymer with a —OH group on the terminus of the PEG chain.
FIG. 24C depicts a synthetic route whereby termini of the polymers shown in FIGS. 24A and 24B can be activated to form a terminal amine, providing a route for coupling to an antigen.
FIG. 25A depicts a route for pyridyl disulfide activation of the polymer chain termini on nanoparticle and micelle surfaces, and thus provides a facile means toward coupling of antigens that possess a free cysteine or are engineered to possess a free cysteine residue.
FIG. 25B depicts a route for protecting thiols as an intermediate conjugation step.
FIG. 25C depicts a conjugation scheme for reacting free thiols to bind moieties to a nanoparticle. Note that NSVYWFFVWL (SEQ ID NO:1) is used as an example.
FIG. 26 depicts a route for pyridyl disulfide activation of a cysteine residue or other thiol residue on the antigen led to SIINFEKL (SEQ ID NO: 2) immobilization as well as appropriate display in MCHI after injection and trafficking to the lymph node, followed by uptake in lymph node-resident DCs.
FIG. 27 depicts a synthetic scheme for derivatization of the N-terminal alpha-amine to a function that can be coupled to a nanoparticle surface via a pyridyl disulfide or a vinyl sulfone moiety, for example. “Flic” represents a characteristic protein, and “NP” represents a characteristic nanoparticle surface.
FIG. 28 is a bar graph that shows how polymer micelles, as described above, were either exposed to serum or not and were then exposed to BMDCs in vitro, after which DC activation was determined by CD86 measurement by flow cytometry. Comparative results with crosslinked core PPS nanoparticles (bearing surface HO— and HS— groups) (NPHS040) and with activation by CpG oligonucleotides is shown. The PEG-PPS polymer micelle implementation is shown to be capable of full activity.
FIG. 29 depicts how danger signals, here illustrated by CpG DNA, can also be conjugated to nanoparticle and micelle surfaces.
FIG. 30 depicts branched 8-arm PEG that activates DCs in vivo. Flow cytometry histograms showing CD11c+DCs isolated from mouse lymph nodes at 24 h after intradermal injection with of either 8-arm PEG, 25 nm polyhydroxylated-NPs with thiols (OH—SH—NP). 8-arm PEG induces upregulation of the DC maturation markers CD86, CD80, and CD40 similar to that of OH—SH—NPs. Histograms are also shown from mice that received injections of negative and positive controls, PBS and LPS, respectively; demonstrating that activation of 8-arm is also comparable to activation with LPS.
FIG. 31 depicts a synthetic scheme by which to form a branched PEG, containing terminal HO-groups for complement activation as well as a site for coupling of an antigen via disulfide exchange at the pyridyl disulfide site.
FIG. 32A shows a synthetic scheme by which to form PEG-PPS-PEI, which forms micelles that can further form a complex with DNA.
FIG. 32B shows results of experiments with plasmid DNA complexes with mixed micelles formed from PEG-PPS blended with PEG-PPS-PEI, which generated ca. 30 nm diameter DNA-containing nanoparticles, which transfected lymph node-resident cells to a high extent; T cells, B cells, and DCs were transfected.
FIG. 33 shows nanoparticle-incorporated immunosuppressant molecules can be used to prevent DC activation after exposure to even very potent DC activation signals such as LPS.
FIG. 34A shows, as a model of a peptide or protein antigen, a transmission electron micrograph of PEG-ss-PPS vesicles encapsulating calcein.
FIG. 34B shows that the calcein of FIG. 34A was efficiently taken up into cellular cytoplasm, with calcein fluorescence being measured as it is de-quenched after vesicle disruption.
FIG. 35 PPS-core nanoparticles, 30 nm, were introduced with mild pressure (A), using a blunt-tipped cannula, to the sublingual tissue in the rodent, and penetrate the tissue very well compared to application without pressure (B).



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Introduction to Invention
Nanoparticles with suitable properties can be used to specifically target therapeutic substances to antigen-presenting cells (APCs) including dendritic cells (DCs) in lymph nodes. The effects of nanoparticle size on lymphatic uptake, lymph node retention, and internalization by lymph node APCs and DCs are demonstrated herein in response to intradermal injections in mice (which have similar size lymphatic capillaries as those in humans—10-80 μm—although these are highly variable in both species [20, 42]). While a variety of nanoparticle sizes may be used, nanoparticles of about 20 nm diameter are most readily taken up and, further, are retained in the lymph nodes for longer times (up to 120 h) than has been previously reported for other particles [31-34, 36].
Certain nanoparticle surface chemistries are surprisingly demonstrated to activate complement and a Th1-mediated immunological response that involves toll-like receptor TLR4 (Examples 18 and 20). These nanoparticles cause surprisingly efficient and powerful activation of the immune system and can be used to make nanoparticle vaccines. The nanoparticles can act as adjuvants with antigens being introduced in or on the nanoparticles, or in a mixture with the nanoparticles; further options are discussed in detail below. The nanoparticles can also be prepared to have surprisingly low toxicities, with the nanoparticles causing essentially no production of tumor necrosis factor alpha (TNF-alpha) or interleukin-6 (IL-6) (Example 19).
A surprising synergy was achieved by combining hydroxyl and thiol groups into the nanoparticles (Example 21). This combination was even more effective than only-hydroxyl preparations. Certain embodiments of nanoparticles thus include combinations of thiols and hydroxyls.
Within the lymph node, it was shown that the complement-activating nanoparticles are internalized effectively by resident APCs (MHCII+ cells which include DCs and some macrophages) and other non-antigen presenting macrophages (MHCII−) without a targeting ligand. A large fraction (up to 50%) of lymph node resident DCs internalized the 20 nm complement-activating nanoparticles, with the number increasing over time. It was found that DC maturation occurred following complement-activating nanoparticle internalization.
Size and surface chemistry are simultaneously important: complement activating nanoparticles that are not sized to enter lymphatics at a given efficiency (e.g., 100 nm nanoparticles) are not as powerful an adjuvant as relatively smaller nanoparticles (e.g., 25 nm) of the same chemistry that have a higher efficiency of entry. Complement non-activating nanoparticles that are sized to readily enter the lymphatics (e.g., 25 nm nanoparticles with methoxyl surface groups) are not as powerful an adjuvant as the same sized nanoparticles of a surface chemistry that does activate complement (e.g., 25 nm nanoparticles with hydroxyl surface groups). That complement activation plays a key role in the mechanism is evidenced by poor immune response in C3−/− animals injected with antigen-coupled 25 nm nanoparticles with methoxyl surface groups. Small (e.g., 20-45 nm) hydroxylated nanoparticles thus offer a strategy to deliver immunotherapeutic agents to DCs and other APCs in lymph nodes. In general, nanoparticle sizes refer to the maximum dimension of the particle, e.g., the diameter of a sphere or the longest length that can be traced through a non-spherical particle, e.g., a rod or ellipse.
Certain surface chemistries can be utilized to activate APCs, including DCs and then induce T cell dependant adaptive immune responses. Some material surfaces can activate the complement cascade, including hydroxylated surfaces [117] or hydroxylated surfaces obtained by stabilization with PLURONIC [118]. Materials surfaces can be conjugated to certain hydroxylated molecules and biomolecules to activate complement. For instance, artisans may apply techniques set forth herein to accomplish the conjugation. Moreover, implants, medical devices, or other carriers besides nanoparticles may receive a layer of complement-activating polymer as described herein, or the polymers or the hydroxyls can be introduced directly onto such materials. In adjuvant development, the conventional approach utilized by scientists to activate cells, such as, DCs, through exogenous activators of the Toll-like receptors, such as lipopolysaccharide (LPS). [119-121]. But it has been discovered that certain nanoparticle surface chemistries can activate a different aspect of innate immunity, namely the complement cascade: the detailed examples herein demonstrate that hydroxylated and/or nanoparticles can activate endogenous components of the complement cascade and that this can in turn activate APCs including DCs and induce T cell dependant humoral and cellular immunity. Other embodiments are nanoparticles with alternative schemes for presenting hydroxyls or nanoparticles with thiols or both thiols and hydroxyls. In other work on nanoparticles as adjuvants, not employing a complement mechanism, polymer nanoparticle size determined the extent to which DCs were targeted and activated: with carboxylated polystyrene nanoparticles, intermediate (45 nm) sized nanoparticles are taken up by DCs and activate them, but smaller ones (20 nm) are not [116]. With nanoparticles described herein, however, complement can be vigorously activated and this provides a powerful signal for activation of DCs and induction of T cell dependant humoral and cellular aspects of the adaptive immune response.
Complement activation is known to enhance the adaptive immune response, specifically B cell immunity. Previous work has demonstrated that complement proteins C3b and C3d can be utilized as a molecular adjuvant for enhancement of B cell-dependant humoral immunity. Immunization of mice with a fusion of C3b or C3d to model antigens demonstrates a significant increase in the acquired immune B cell response compared to free antigen alone [134, 135]. The mechanism that C3b and C3d adjuvants work may be through direct binding of the C3d receptor (CD21/35) which associates with CD19, a known amplifier of B cell activation. However, CD21/35 has been found to not always be necessary for this B cell response [136]. One certainty to C3b- and C3d-antigen fusions is that their adjuvant capacity for humoral immunity is through direct interaction with B cells [137]. This is different than T cell-dependant humoral immunity, which occurs when antigen is uptaken by DCs as taught herein, DCs mature, DCs process and present antigen through MHC II to CD4 T cells, CD4 T cells present antigen to B cells, and finally B cells produce antibodies. While complement has been discovered to be involved in T cell-dependant immunity, the mechanisms by which this occurs have not been described [138]. Moreover, it has not been previously suggested that complement activation could be used as a molecular adjuvant for T cell dependant immunity.
The systems herein, however, describe how complement activation can be used as a molecular adjuvant for T cell dependant immunity. Moreover, some embodiments include nanoparticles that activate complement through nanoparticle surface chemistries. Specifically, for instance, the results show that hydroxylated and/or thiolated nanoparticles induce DC maturation and demonstrate for the first time that complement activation through hydroxylated and/or thiolated surfaces can be utilized as a danger signal to induce DC maturation. Also as described herein, complement-activation through hydroxylated and/or thiolated nanoparticles is used as an adjuvant for the induction of DC-mediated T cell-dependant humoral and cellular immunity.
Immune System Targeting
Antigen-presenting cells (APCs) are highly efficient phagocytic cells that utilize MHC class I, II and other co-stimulatory molecules (i.e., CD86 and CD80) to stimulate naïve T cells and induce cell-mediated immunity. APCs, which include some macrophages and the more potent dendritic cells (DCs), are present in peripheral tissues where they act as sentinels that, following internalization and processing of foreign antigens, subsequently undergo maturation and migration to lymph nodes for the purpose of antigen presentation to T cells [1-3]. With the critical roles that APCs and DCs play in adaptive immunity, various experiments are being made to target these cells with immunomodulating agents, such as DNA, proteins, and polypeptides [4-14]. Polypeptide is a term that refers to two or more amino acids joined together, and includes proteins.
Polymer- and liposome-based delivery systems have focused primarily on delivery of protein and DNA to peripheral DCs, where they first internalize the drug vehicles and then migrate to lymph nodes within  ̃1-2 days to activate T cells [9, 12, 13]. Until recently, it was not clear whether immature DCs, capable of taking up antigens, were present in lymph nodes. However, recent studies have established that a substantial fraction of resident DCs in the lymph nodes are phenotypically immature and capable of internalizing antigens and particles there [15, 16]. Thus, as explained herein, resident lymph node APCs may also be utilized as targets for immunotherapeutic drugs or other therapeutic agents. A potential benefit of targeting lymph node APCs or DCs instead of those in peripheral sites is that premature antigen presentation (i.e., a migrating DC that expresses antigen on its surface prior to reaching a lymph node) can often lead to immune cell tolerance [13, 17, 18]; therefore, delivery to lymph node APCs may potentially avert this problem. Additionally, other DC targeting studies use conjugated targeting ligands such as anti-Dec-205 and anti-CD11c to increase DC specificity [4, 5, 8, 9, 12, 19].
What has not been conventionally appreciated, however, is that one can effectively exploit the fact that DCs are by nature highly phagocytic and present in lymph nodes at high concentrations. Accordingly, materials and methods to specifically target these cells in lymph nodes have been developed, as explained herein, including targeting without the use of a targeting ligand. A targeting ligand refers to a chemical group that specifically binds to a particular chemical group on a cell, e.g., a cell surface receptor or a cell surface protein. Thus some embodiments can be targeted based on the size and other physical properties and are targeted with no exogenous polypeptide, with no exogenous ligand, with no exogenous nucleic acid, with no antibody or fragment thereof, or with no exogenous ligand for any of: a receptor, cell surface molecule, extracellular matrix molecule, cell surface antigen, cell marker molecule, or polysaccharide.
In order to target APCs, including DCs, in lymph nodes, it is useful, as demonstrated herein, to design delivery vehicles that can be readily taken up into lymphatic vessels and retained in draining lymph nodes. A delivery vehicle refers to an agent, e.g., a particle or micelle that delivers a therapeutic agent, e.g., an antigen or immunosuppressant drug. A primary role of the lymphatic system is the uptake of fluid and particulates from the interstitial space as a small but important component of the microcirculation [20-23].
Other in vivo lymphatic targeting experimental studies using liposomes and polymer particles to investigate the lymphatic system have indicated that particle size can be a factor for lymphatic uptake from the interstitial space [21, 24-29]. Liposomes larger than 170 nm generally showed poor lymphatic uptake and remained at the injection site, whereas particles in the range of 40-70 nm showed significant uptake into lymphatic vessels [25].
One such study using carboxylated polystyrene particles teaches that particles only in the narrow range of 40-50 nm are practically useful because this size is a danger signal recognized by DCs; consequently, DCs are activated in dermal sites and not in lymph nodes [116]. This polystyrene bead study showed that beads accumulate in lymph nodes at intermediate sizes (40 nm) more than smaller (20 nm) and larger (>100 nm) sizes and taught that 40-50 nm was the size that should be used for beads. More specifically, this study showed that very small particles (20 nm) and larger particles (100 nm) were found to accumulate significantly less than 40 nm particles in cells that were positive for DC markers, as indicated by the DC antigen DEC205 [116]. The authors teach that 40 nm polystyrene beads cause activation and migration of DCs in dermal sites to lymph nodes; therefore 40 nm beads could not be targeting lymph node resident DCs.
Further, the polystyrene bead study teaches that bead size is a danger signal for DCs because DCs have evolved to recognize viral size ranges. The bead size, therefore, would control successful DC targeting, with correctly sized beads being recognized by DCs in the periphery, and causing activation of the DCs. This teaching is in sharp contrast to the successful use of smaller nanoparticles for DC activation (less than about 40 or about 35 or about 25 nm) as described herein. This teaching is also in contrast to the results herein showing that surface chemistry is a danger signal, e.g., as in nanoparticles which utilize hydroxyl and/or thiolated surface chemistry to activate complement as a danger signal. Moreover results herein relate particle size to lymph node targeting capability and not DC size recognition. For instance, the, 25 nm PLURONIC-stabilized complement-activating nanoparticles were better than 100 nm PLURONIC-stabilized complement-activating nanoparticles at activating DCs and adaptive T cell immunity after in vivo injection.
The carboxylated polystyrene beads were used as an experimental model system at least in part because their convenient synthesis and emulsion polymerization characteristics give rise to a narrow and controllable size distribution [116]. Potential disadvantages for use as a therapeutic or prophylactic system are associated with the polystyrene beads. For example, no biological pathway exists by which such particles may be degraded and eliminated from the body. In contrast, a biodegradable system as described herein will degrade readily and effectively to soluble polymer in response to an in vivo environment, e.g., as in PPS nanoparticles that degrade under oxidative conditions encountered after endocytosis and processing. While degradation of the nanoparticles may be beneficial, it is not necessarily a requirement for use as an adjuvant.
Interactions between the particle surface and the interstitium may be another factor that plays a role in lymphatic uptake [30]. Steric stabilization by coating liposomes and particles with hydrophilic layers such as poly(ethylene glycol) (PEG) and its copolymers such as PLURONICS (including copolymers of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)) may reduce the non-specific interactions with proteins of the interstitium as demonstrated by improved lymphatic uptake following subcutaneous injections [21, 27, 31-35]. All of these facts point to the significance of the physical properties of the particles in terms of lymphatic uptake.
However, while smaller particles are taken up more readily, they also pass out of the lymph node more easily; achieving both efficient lymphatic uptake and lymph node retention is thus significant. Accordingly, certain nanoparticle embodiments have features that address both uptake and retention, as is evident from the examples and embodiments set forth herein. With respect to the size of nanoparticles, for example, investigations of the lymphatic system indicated that only 1-2% of injected 70 nm liposomes are retained in the lymph nodes beyond 12 h post-injection [30], and that node retention of large liposomes (>70 nm) is more efficient than that of smaller liposomes [24, 29]. This apparently is due in part to the fact that lymph node macrophages phagocytose larger particles more efficiently. While it is conventionally assumed that coating of liposomes with steric protectants such as PEG should reduce phagocytosis by macrophages, it has been shown that such coating does not significantly affect lymph node retention [36]. It has also been conventionally assumed that particles phagocytosed in the lymph nodes are done so primarily by macrophages [21, 27, 29, 30, 32, 36, 37]. With carboxylated polystyrene nanoparticles, it was found that 20 nm nanoparticles were taken up much less by DCs than were 40 nm nanoparticles in vivo [116]. Therefore, besides delivering drugs for uptake by macrophages, it is advantageous to deliver drugs to lymph nodes for uptake by other APCs, including DCs. As shown below, certain embodiments of the particles of this invention are, in fact, taken up by APCs and/or DCs even at small sizes at least as low as 20 nm.
Previous studies have suggested that following antigen uptake, powerful biological “danger signals” such as inflammatory cytokines (i.e. CD40 ligand) are necessary to mature DCs and subsequently induce cell-mediated immunity [5, 12, 38]. However it may be advantageous to forego such signals. In fact, in some embodiments, it is the nanoparticles themselves are used as a maturation stimulus that avoids the use of conventional biological biomolecule “danger signals”, e.g., some polypeptides, antibodies, nucleic acid sequences. These results are evident in the maturation response of DCs that was observed following nanoparticle internalization in vivo.
Nanoparticle Formulations
As documented herein, size is related to nanoparticle uptake and retention in lymph nodes. Nanoparticle lymphatic uptake, lymph node retention, and localization within lymph nodes and among cell populations there are documented herein. One challenge using conventional approaches is obtaining both efficient lymphatic uptake and lymph node retention, since nanoparticle properties, such as size and surface characteristics, can have conflicting effects. In general, smaller particles have better lymphatic uptake than larger particles but lower lymph node retention. Nanoparticles with a size of about 5 nm to about 100 nm diameter are preferred; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 nm. The nanoparticles may be made in a collection that of particles that has a mean diameter from about 5 to about 100 nm; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., from about 10 to about 70 nm. The size distribution of such a collection of particles can be controlled so that a coefficient of variation (standard deviation divided by mean particle size) around a mean diameter of a collection of the particles may be less than about 50, about 35, about 20, about 10, or about 5 nm. [39]; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated.
Physical properties are also related to a nanoparticle's usefulness after uptake and retention in lymph nodes. These include mechanical properties such as rigidity or rubberiness. Some embodiments are based on a rubbery core, e.g., a poly(propylene sulfide) (PPS) core with an overlayer, e.g., a hydrophilic overlayer, as in PEG, as in the PPS-PEG system recently developed and characterized for systemic (but not targeted or immune) delivery [39, 40]. The rubbery core is in contrast to a substantially rigid core as in a polystyrene or metal nanoparticle system. The term rubbery refers to certain resilient materials besides natural or synthetic rubbers, with rubbery being a term familiar to those in the polymer arts. For example, cross-linked PPS can be used to form a hydrophobic rubbery core. PPS is a polymer that degrades under oxidative conditions to polysulfoxide and finally polysulfone [41], thus transitioning from a hydrophobic rubber to a hydrophilic, water-soluble polymer [39]. Other sulfide polymers may be adapted for use, with the term sulfide polymer referring to a polymer with a sulfur in the backbone of the mer. Other rubbery polymers that may be used are polyesters with glass transition temperature under hydrated conditions that is less than about 37° C. A hydrophobic core can be advantageously used with a hydrophilic overlayer since the core and overlayer will tend not to mingle, so that the overlayer tends to stericly expand away from the core. A core refers to a particle that has a layer on it. A layer refers to a material covering at least a portion of the core. A layer may be adsorbed or covalently bound. A particle or core may be solid or hollow. Rubbery hydrophobic cores are advantageous over rigid hydrophobic cores, such as crystalline or glassy (as in the case of polystyrene) cores, in that higher loadings of hydrophobic drugs can be carried by the particles with the rubbery hydrophobic cores.
Another physical property is the surface's hydrophilicity. A hydrophilic material has a solubility in water of at least 1 gram per liter when it is uncrosslinked. Steric stabilization of particles with hydrophilic polymers can improve uptake from the interstitium by reducing non-specific interactions; however, the particles' increased stealth nature can also reduce internalization by phagocytic cells in lymph nodes. The challenge of balancing these competing features has been met, however, and this application documents the creation of nanoparticles for effective lymphatic delivery to DCs and other APCs in lymph nodes. Thus some embodiments include a hydrophilic component, e.g., a layer of hydrophilic material. Examples of suitable hydrophilic materials are one or more of polyalkylene oxides, polyethylene oxides, polysaccharides, polyacrylic acids, and polyethers. The molecular weight of polymers in a layer can be adjusted to provide a useful degree of steric hindrance in vivo, e.g., from about 1,000 to about 100,000 or even more; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., between 10,000 and 50,000. The examples include a particle with a hydrophilic surface that is a PEG derived from a PLURONIC that was used as a stabilizer during synthesis as an emulsion.
The nanoparticles may incorporate functional groups for further reaction. Functional groups for further reaction include electrophiles or nucleophiles; these are convenient for reacting with other molecules. Examples of nucleophiles are primary amines, thiols, and hydroxyls. Examples of electrophiles are succinimidyl esters, aldehydes, isocyanates, and maleimides. For instance, using PPS-PLURONIC nanoparticles as an example, particles are then synthesized as described in Example 1 with the difference that 2% of the PLURONIC is replaced with OVA/OVA257-264/OVA323-339 derivatized PLURONIC. A total amount of 1.5% PLURONIC is used. Reaction time is reduced to 2 h and base is added at 1:1 equimolar ratio to initiator-thiols in order to reduce the exposure of the protein or peptides to basic conditions during nanoparticle synthesis. This scheme is only one exemplary method for PEG functionalization; several other approaches can be utilized depending on the protein or peptide being conjugated [111].
The nanoparticles may also incorporate functional groups or motifs for complement activation. A preferred functional group is hydroxyl, which is particularly effective for activating complement as documented herein. Other functional groups that are nucleophilic can react with the thioester in C3. It is demonstrated herein that hydroxylated nanoparticle surfaces are particularly useful in targeting APCs, including DCs, in the lymph node. In the case of the PPS nanoparticles of the examples herein, hydroxylation was obtained by stabilization with PLURONIC terminated with hydroxyl groups. When these hydroxyl groups were converted to methoxy groups to block the hydroxy group, the nanoparticles did not function well as adjuvants. When the hydroxylated nanoparticles were tested in C3−/− mice, their adjuvant effect was vastly diminished. These results, combined with measurements described in the examples herein of C3 activation, demonstrate the particular usefulness of targeting APCs, including DCs, with complement-activating nanoparticles. Accordingly, in some embodiments, nanoparticles rely only on OH to activate complement and exclude one or more of: cations at pH 7.0-7.4, amines, primary amines, secondary amines, anions at pH 7.0-7.4, thiols, zwitterions at pH 7.0-7.4; alternatively such groups are present on the nanoparticle but it is a layer of polymer on the nanoparticle that excludes one or more of such groups. Alternatively, the nanoparticle and/or layer has no group effectively capable of forming an ion at pH 7.0 to 7.4 except OH.
Functional groups can be located on the particle as needed for availability. One location can be as side groups or termini on the core polymer or polymers that are layers on a core or polymers otherwise tethered to the particle. For instance, examples are included herein that describe PEG stabilizing the nanoparticles that can be readily functionalized for specific cell targeting or protein and peptide drug delivery.
Biodegradable polymers may be used to make all or some of the polymers and/or particles and/or layers. Biodegradable refers to polymers that are subject to degradation by spontaneous hydrolysis, chemical attack by enzymes that cleave specific amino acid sequences, or by incorporating functional groups that are oxidation-sensitive. Polymers subject to spontaneous hydrolysis will degrade in vitro in aqueous solution kept at a pH of 7.0 to 7.4 as a result of functional groups reacting with the water in the solution. The term “degradation” as used herein refers to becoming soluble, either by reduction of molecular weight (as in the case of a polyester) or by conversion of hydrophobic groups to hydrophilic groups (as in the case of PPS). Polymers with ester groups are generally subject to spontaneous hydrolysis, e.g., polylactides and polyglycolides. Many peptide sequences subject to specific enzymatic attack are known, e.g., as degraded by collagenases or metalloproteinases: sequences that are degraded merely by biological free radical mechanisms are not specifically degraded. Polymers with functional groups that are oxidation-sensitive will be chemically altered by mild oxidizing agents, with a test for the same being enhanced solubilization by exposure to 10% hydrogen peroxide for 20 h in vitro. For example, PPS is an oxidation-sensitive polymer [39].
While PPS particles were used as an exemplary system in certain embodiments to demonstrate how to make and use the nanoparticles, alternative materials may be used. In general, the features for each component of the particle system may be freely mixed-and-matched as guided by the need to make a functional particle. For instance, other particles that are small (e.g., less than about 100 nm or less than about 70 nm) and thus enter the lymphatic circulation efficiently may be used. Such particles may optionally be grafted with an overlayer of PEG, or otherwise incorporate a hydrophilic polymer, and may optionally be grafted with antigen, danger signals, or both. For example, a PEG-containing block copolymer can be adsorbed to a degradable polymer nanoparticle of appropriate size, and antigen can be further attached to the surface of such a treated polymer. As another example, a PEG-containing block copolymer can be adsorbed to an inorganic nanoparticle of appropriate size, and antigen can be further attached to the surface of such a treated polymer. While degradation of the nanoparticle core may be desirable, it is not necessarily required.
Micellar systems may also display the same useful characteristics as described above, including micelles formed from AB and ABA block copolymers of poly(ethylene glycol) and PPS [100-104]. When such copolymers are formed with a molecular fraction of poly(ethylene glycol) that is relatively high, e.g., in excess of about 40%, then spherical micelles can be expected to form under certain conditions. These micelles can be small, e.g., meeting the size mentioned above for lymphatic entry, and may optionally be grafted with an overlayer of PEG, or otherwise incorporate PEG or other polymers to achieve similar properties. Moreover, they can be conjugated with antigen, as taught herein, danger signals or both at the micelle surface. The block copolymer can terminate in a hydroxyl group, for complement activation, and it is particularly beneficial to have the hydrophilic block terminate in a hydroxyl group, so that this hydroxyl group will be more readily available on the micellar nanoparticle surface for complement binding. Such hydroxylated such surfaces can be tailored to effectively activate complement. A particularly useful hydrophilic block is PEG, terminated in a hydroxyl group. In addition to micelle-forming polymer architectures, block sizes and block size ratios can be selected to form vesicular structures. There also exists a number of other possible chemical compositions of micellar formulations that may be used [105-108].
Polymer micelles may be advantageous from the perspective of production of very small structures with very narrow size distributions, as well as from the perspective of faster elimination from the body than crosslinked core nanoparticles. This feature was demonstrated with polyethylene glycol-poly(propylene sulfide) (PEG-PPS) nanoparticles made as micelles capable of displaying surface hydroxyl groups as well as surface thiol groups, e.g., see Example 23 One route to synthesis of hydroxyl-PEG-PPS with or without blockade of a terminal —SH group is shown in FIG. 22, and one route to synthesis of methyl-PEG-PPS is shown in FIG. 23.
Moieties may be attached to nanoparticles or branched polymers, e.g., antigens, peptides, antibodies, antibody fragments, nucleic acids, nucleic acid fragments. Examples of attachment of moieties to micellar nanoparticles are described below and depicted in FIGS. 24-27, and 29 see also Example 23. FIG. 28 summarizes the results of detailed experiments showing that nanoparticles prepared as micelles are comparable to crosslinked-core nanoparticles; the bar labeled NPHS040 is a PPS crosslinked-core nanoparticle of hydroxyl and thiol surface groups.
In some embodiments, micellar nanoparticles are prepared and dried to preserve the micellar structure, these are referred to as “dried micelles” recognizing that micelles is a term that generally refers to structures in a solution. One method of forming dried micelles is to freeze a collection of micelles and lyophilize them to remove the water. The resultant materials may be kept at temperatures less then 0° C. until use or may be stored at higher temperatures. The dried micelles may also be stored dry after other drying processes, such as vacuum evaporation or spray drying. The dried micelles may be administered dry or may be re-hydrated before administration. Alternatively, micelles may be frozen in a solution and administered in a frozen state or thawed before use, e.g., by adding a room temperature physiologically acceptable solution.
Some polymer systems are themselves nanoparticulate and are included in the term nanoparticle. For example, dendrimers are a class of polymer that can be nanoparticulate in the 10 nm range [141]. These polymers comprise a high number of functional groups at their surface, for example which have been used to conjugate to biomolecules and other groups [142, 143]. Analogously, antigen could be conjugated to the dendrimer surface. Moreover, the functional groups on the dendrimer surface could be optimized for complement activation, for example by hydroxylation. Some dendrimer-DNA complexes have been demonstrated to activate complement [144, 145]. Thus, dendrimers represent an interesting nanoparticulate chemistry that could be adapted for lymphatic targeting using the techniques described herein, for antigen conjugation, and for complement activation, e.g., as in U.S. Pat. Pub. Nos. 2004/0086479, 2006/0204443, and in U.S. Pat. Nos. 6,455,071 and 6,998,115, which are hereby incorporated by reference herein to the extent they do not contradict what is explicitly disclosed.
Dendritic molecules are repeatedly dividing polymers in which the atoms are arranged in many arms and subarms radiating out from a central core. Dendrimers are characterized by their structural perfection as based on the evaluation of both symmetry and polydispersity and require particular chemical processes to synthesize. Accordingly, an artisan can readily distinguish dendrimer nanoparticles from non-dendrimer nanoparticles. Embodiments of nanoparticles herein include non-dendrimer nanoparticles. Some non-dendrimers are nanoparticles that are branched polymers that have branches with a degree of polymerization of at least 5, with the term branch referring to a polymer chain portion located between branch points. Some non-dendrimers have branches with a molecular weight of at least 200, e.g., a polyethylene glycol with a degree of polymerization of at least 5.
Dendrimers have a shape that is highly dependent on the solubility of its component polymers in a given environment, and can change dramatically according to the solvent or solutes around it, e.g., changes in temperature, pH, ion content, or after uptake by a DC. In contrast, nanoparticles that have physical dimensions that are relatively more stable than dendrimers or other merely branched polymer systems can be useful for storage purposes or as related to or biological activity, e.g., a solid core with a hydrophilic corona will consistently present the corona to its environment. Accordingly, some embodiments of nanoparticles rely on particles that are not dendrimers, or that have a core that is a solid and/or have a core that is a cross-linked hydrogel. A PPS-based nanoparticle is not a dendrimer and has a solid core.
Embodiments include very small nanostructures displaying a moiety as described herein that is capable of activating complement. Such structures may also display an antigen or other moiety. Certain embodiments are water-soluble multifunctional polymers, such as branched polymers, for instance, branched PEGs having a branch or a plurality of branches with a degree of polymerization of at least about 5. Water soluble is a term that refers to materials having a solubility of at least 1 gram per liter of water. FIG. 30 (see Example 24) shows that an 8-arm (8 termini) branched PEG (total MW about 40,000) with hydroxyl end groups effectively activated dendritic cells in vivo. FIG. 31 provides a detailed scheme for making branched PEGs. A terminus refers to a polymer end groups, with the 8-arm branched PEG having 8 hydroxyl termini. A branch refers to a side chain extending from a polymer backbone or core. In the case of symmetrical polymers, the core is the central portion of the molecule. The term backbone is customary in these arts and its metes and bounds will be immediately appreciated by artisans.
Accordingly, embodiments include water soluble polymers with a molecular weight of between 10,000 and 1,000,000 and between four and 5,000 termini, with the polymers activating dendritic cells in vivo, e.g., as tested using methods of Example 24 and/or FIG. 30. Artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., at least four termini, or at least 8 termini, or 4-20 termini. The polymer is sized to penetrate through interstitial tissue to enable it to be collected within the draining lymphatics, enabling targeting of APCs resident there. As such, a spherical branched polymer may be preferable to a long branched polymer of same molecular weight and number of termini, in that longer polymers may more easily become entangled within the protein and glycosaminoglycan network of the tissues. Nevertheless, so long as penetration through the interstitial tissue is adequate, long polymers with side branches are useful. As such, embodiments include both branched polymers emanating from one or several central branch points, as well as long polymers with side branches. The branched polymers may be non-dendritic polymers.
As per FIG. 31, branched polymers may further contain functional groups for further reactions, e.g., amines or thiols. Also, other moieties may be attached as described herein, e.g., antigens or danger signals. Moreover, the polymers may be made with biodegradable groups. Various embodiments set forth herein describe uses and modifications of nanoparticles; these uses and modifications may also be applied to water soluble dendrimers or branched materials as described herein.
Nanoparticles may also be made as vesicles, e.g., a watery-core vesicle. The watery-core vesicles may be made with amphiphilic copolymers, for instance, and processed to achieve a nanoparticulate structure and dimensions. The vesicles may be made with a reduction-sensitive linkage. The vesicles may be made in association with antigenic or therapeutic moieties.
Immunotherapy with Nanoparticles
In use, such nanoparticles are useful as, e.g., antigen and drug delivery vehicles to target APCs, specifically DCs, in lymph nodes. The ability to deliver antigen and/or drugs and/or danger signals to DCs in lymph nodes is a useful approach to immunotherapy. The ability to target lymph node DCs and other APCs effectively with nanoparticles provides a method for delivery of antigenic proteins and polypeptides and antigen-encoding nucleic acids. Since DCs are critically involved in initiating cell-mediated immunity by antigen presentation to T cells, this delivery approach can be utilized for several vaccine and immunotherapy applications. Further, the nanoparticles are useful as diagnostic tools (e.g., imaging), research tools (e.g., as sold in ALDRICH catalogs or for visualization using microscopes), or in vitro drug delivery or visualization (e.g., APC and/or DC and/or macrophage uptake in vitro of drugs or imaging agents).
The antigen and/or drugs and/or danger signals may be covalently attached to the particles, adsorbed to the particles, loaded into the particles, or mixed with a collection of the particles for contemporaneous introduction into a patient. Motifs for covalent bonding are discussed elsewhere herein, and may be applied in this instance also. Adsorption can be accomplished by mixing the agent and the particles for a predetermined amount of time and then physically separating the particles from the mixture, e.g., by centrifugation or filtration. The separation may take place before or after administration, in vitro or in the body, e.g., by injecting the mixture and allowing diffusive and convective forces to separate the components. Loading may be performed during or after particle synthesis. For instance, the particles may be polymerized in the presence of the agent, which will become entrapped, either by adsorption or by phases separation as in a micellar-based polymerization. Loading after synthesis may be accomplished as needed, e.g., by exposing the particles to an agent in a first solvent that swells the particles or permits ready diffusion of the agent and then exposing the particles to a second solvent that contracts the particles or restores them to an aqueous solution that stops or slows diffusion of the agent, for instance: loading a hydrophobic drug in organic solvent and storing the particles in aqueous solution. Mixing for contemporaneous introduction may be accomplished by, e.g., introducing the particles into a syringe having a solution of antigen and/or drugs and/or danger signals and coinjecting them into a patient.
Nanoparticles, including micelles, or branched polymers as described herein may include functional groups for the activation, e.g., hydroxyls and/or thiols. The materials may also be prepared with further functional groups for further derivatization, e.g., amine, thiol, unpaired cysteine residue. In some embodiments, collections of nanoparticles or branched polymers are prepared with functional groups both for immune activation and also for further derivatization. In other embodiments, collections of nanoparticles or branched polymers are prepared with either functional groups for immune activation or functional groups for derivatization, and the collections are administered to a patient separately or as a mixture, or separately but essentially at the same time. The nanoparticles or branched polymers with derivitizable groups may be derivatized with moieties as described herein to make a collection of derivatized materials. Any of the collections may further be used in combination with, or mixed with, adsorbed or soluble agents as described herein, or loaded with agents as described herein. Methods include stimulation of APC or DC maturation at a lymph node with a nanoparticle or branched polymer.
Nanoparticles, including micelles, or branched polymers may be prepared to activate a full response of the adaptive immune system, for instance see Examples 18-21. Methods include administering the materials in an amount effective to stimulate a Th1 biased response and/or IL-12 secretion and/or a TLR4-mediated immune response in an APC or DC at a lymph node. Methods include stimulating an in vitro or in vivo immune response (full adaptive immune response, complement activation, antibody production, IL-12 secretion, APC maturation, DC maturation, CD80-induction), optionally without production of, or essentially without (meaning not detectable in a blood sample of a patient with assays commonly used for these molecules) TNF-alpha and/or IL-6 production.
Immunotherapy with Nanoparticles and Immunosuppressive Drugs
Immunosuppression is a critical form of immunotherapy that is greatly needed in situations of clinical transplantation (e.g., allografts) and autoimmune diseases (e.g., multiple sclerosis). The use of immunosuppressive drugs such as corticosteroids (e.g., Cyclosporin A) and rapamycin has led to great advances in the treatment of these immune disorders [122]. T cells are generally considered to be important targets of immunosuppressive drugs, which act by inhibiting genes for inflammatory cytokines, primarily IL-2 and TNF-γ, and therefore reduce T cell proliferation. Another approach for immunosuppression being developed is the use of antibodies to block the T cell receptors CD28 and CD40 [123]. Blocking these receptors leads to insufficient activation by co-stimulatory molecules located on DCs and therefore causes a tolerance effect that effectively aborts T cell proliferation. However treatment with corticosteroids or blocking antibodies is very non-specific and may lead to side effects such as reducing the ability of the immune system to fight off other infections. Therefore a strategy for more specific immunosuppression, one that induces antigen-specific tolerance would be an exceptional advance in immunotherapy.
Recently it has been discovered that DCs may be a target for immunosuppression; in addition to their ability to stimulate T cell immunity, DCs are also capable of regulating T cell tolerance [124]. Dexamethasone (Dex) is a synthetic glucocorticoid utilized for immunosuppression in applications such as preventing rejection of allograft transplants. Traditionally it has been thought that glucocorticoids act solely on T cells. However recent studies have demonstrated that Dex can act on DCs to down regulate the expression of the co-stimulatory molecules and the secretion of inflammatory cytokines [125,126]. This has substantial implications for the potential to utilize DCs for the induction of tolerance. A DC that presents antigen to T cells in the absence of co-stimulatory molecules will induce T cell anergy or tolerance to the presented antigen.
Immunosuppressive drugs may be delivered with the nanoparticles. In some embodiments, antigen can be effectively targeted to the lymph node DCs by virtue of their size and interaction with interstitial flows and entry into the lymphatics. Delivery of an antigen and at the same time as immunosuppressants to prevent DC activation would lead to tolerance. In such cases, nanoparticles that do not activate complement may be beneficial. Some of the major immunosuppressive drugs are glucocorticoids, which are hydrophobic and can be loaded into the hydrophobic PPS core of the nanoparticles. Certain glucorticoids such as Dexamethasone, tacrolimus, and Cyclosporin A have demonstrated that they inhibit the maturation and allostimulatory capacity of DCs by downregulating the expression of co-stimulatory molecules (i.e., CD80 and CD86) and secretion of inflammatory cytokines (i.e., IL-6 and TNF-α) [125-131].
Immunosuppressive drugs may be loaded into nanoparticles described herein and introduced into a patient. The nanoparticles may be specifically targeted to the lymphatic system, and the lymph nodes, and may be specifically targeted for uptake by APCs and/or DCs. Hydrophobic drugs or other agents may advantageously be delivered using nanoparticles with a hydrophobic components or cores. Further, an antigen may be delivered in combination with a drug. The antigen may be associated with a nanoparticle, e.g., by coinjection, adsorption, or covalent bonding.
For example, one strategy is to load hydrophobic immunosuppressive drugs (e.g., Dex) into the core of nanoparticles stabilized with methoxy-terminated moieties (thus not hydroxylated and not complement activating) nanoparticles and graft antigen to the surface. Therefore by delivering Dex or other immunosuppressive drugs along with antigen to DCs in lymph nodes, it is possible to down-regulate co-stimulatory molecules but still deliver antigen and in turn cause antigen-specific tolerance. Therefore nanoparticles, e.g., PPS nanoparticles stabilized with methoxy-terminated PLURONIC, with the combination of antigen conjugation and immunosuppressive drug loading, may be utilized to induce tolerance for applications such as therapy for autoimmune diseases and transplant rejections.
Further, immunosuppressants may be used in combination with schemes to transfect cells, e.g., to make tolerogenic vaccines. A collection of nanoparticles may be made with particles loaded with immunosuppressants and also derivatized to carry DNA or RNA, or separate collections of immunosuppressant-delivering particles may be used in combination (mixture or separate administration) with nucleic-acid delivering particles. Immunosuppressive agents can thus be used in combination with immunotherapy with antigens delivered by nanoparticles, including micelles or non-dendritic polymers.
Immunotherapy with Antigens
APCs that internalize protein antigens can process and present the antigenic peptide epitope through MHC-I and II pathways. One immunotherapeutic approach involves covalently attaching one or more antigens to a nanoparticle, or otherwise associating them. Antigens are polypeptides without or with glycosylation that can be recognized by an immune system, and are generally at least about three amino acids in length. Antigens can also be encoded via nucleic acid sequences such as DNA or RNA. For example, DNA can be encode an antigen if it encodes a polypeptide antigen present on pathogens. Following delivery of the DNA to nuclei of APCs, expression of the antigenic polypeptide antigen would occur and presentation of it on MHC I. Whole proteins can be used, but antigenic fragments can be used, so that polypeptides having between about 3 to about 20 residues can be used; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g. less than about 10 residues. Since nanoparticles may be used that directly activate the complement system without involving a polypeptide, antigens that do not activate the complement system may be used.
Antigens (or other moieties) may also be delivered by attaching the antigen to a branched polymer or nanoparticle, including micelle, via a linker that includes a substrate specifically cleaved by a protease, with the substrate being involved in antigen processing. For instance, the cathepsin substrate KQKLR (SEQ ID NO:3) may be incorporated into a linker and is cleaved by cathepsin S. Antigens may also be attached to the nanoparticle or branched polymer via a disulfide bond, which is reduced after uptake by APCs. For instance, an antigenic peptide may include a cysteine residue or sulfide moiety that is available to form a disulfide bond with a free sulfide on the polymer. FIGS. 25A, 25B, and 25C depict methods of attaching peptides or other moieties to nanoparticles or branched polymers. Experiments show that the peptides are released into the target cells and are bioactive. FIG. 26 shows that the peptide SIINFEKL (SEQ ID NO:2) attached to nanoparticles was immobilized to the particles, delivered to lymph nodes in vivo, was taken up by lymph-node dwelling DCs, and displayed in MHC1 (Example 23). Certain embodiments include nanoparticles or branched polymers that incorporate an antigen or other moiety that is taken up by an APC, DC, or lymph-node resident APC or DC and released to the cell in bioactive form, or released to the cell for expression in an MHC complex. The linkage for the moiety optionally includes a disulfide bridge or an peptidic sequence cleaved by a protease, e.g., an intracellular protease.
Antigens can be used for immunotherapy against many different diseases. One specific application is for tumor immunotherapy. Useful antigens are displayed on tumor cells but not healthy cells. Several antigens have been identified as specific to certain types of tumors, such as Caspase-8, MAGE-1, Tyrosinase, HER-2/neu, and MUC-1 [112]. With this in mind, nanoparticles can be used to deliver such antigens to DCs in lymph nodes as a means for activating T cells to attack tumors. Another application is prophylaxis for infectious diseases. Exposure to antigens can create resistance against such diseases or act as a vaccination for various conditions.
The ability to efficiently transfect cells in the lymph node is also useful for induction of immunity, in the presence of a DC activation signal, or antigen-specific tolerization in the absence of a DC activation signal. In the absence of CpG DNA, transfection may lead to antigen expression, and moreover prolonged antigen expression, in the absence of a danger signal and thus co-stimulatory molecule expression. Accordingly, embodiments include collections of nanoparticles that are free of CpG DNA. Moreover, just as APC-activating hydrophobic immunomodulators can be incorporated, hydrophobic immunosuppressant molecules can also be incorporated, such as dexamethasone, as already discussed. Methods include transfection of cells to express an antigen specifically and directly within the lymph node, with the transfection optionally being accompanied by an immunosuppressant drug. Nanoparticles or polymers described herein that contain both a vector and a drug may be used, or nanoparticles/polymers having one and/or the other may be mixed together or used serially or one or the other may be administered over time, e.g., a dosage of transfection agents and repeated dosages of immunosuppressants.
So-called tolerogenic vaccine formulations have to date been remarkably crude. The ability to deliver antigen to the lymph node directly avoids a quandary in tolerogenic vaccines, namely how to deliver antigen to DCs within the lymph node, but not have those DCs be activated. Normally, if antigen is collected in the periphery, DCs to not migrate to the lymph node unless they are activated: thus the quandary. In certain embodiments, antigen is complexed or conjugated to DC-nonactivating nanoparticles, thereby providing tolerogenic, antigen-specific formulations. Moreover, such nanoparticles, whether formed by inverse emulsion polymerization or by micellization, can be loaded with an immunosuppressant molecule, e.g., as dexamethasone, sirolimus, tacrolimus, everolimus, or Cyclosporine A. These drugs further prevent any DC activation that might pre-exist or that might be induced by the process of nanoparticle uptake. FIG. 33 (see Example 26) shows that bone marrow-derived DCs can be exposed to dexamethasone-loaded nanoparticles, and then can be exposed to very strong DC activation signals such as LPS; the nanoparticle-incorporated immunosuppressant can prevent DC activation even to such a strong signal. Other immunomodulatory molecules, e.g., galectin-1 or iC3b, could also be conjugated to the nanoparticle surface or bound to or within the nanoparticle. Certain embodiments provide nanoparticles or polymers described herein targeted to lymphatic cells that expose the cells to an antigen and/or an immunosuppressant. Nanoparticles or polymers described herein that have both an antigen and a drug may be used, or nanoparticles/polymers having one and/or the other may be mixed together or used serially or one or the other may be administered over time, e.g., a dosage of transfection agents and repeated dosages of immunosuppressants.
Moreover, immunomodulatory drugs may be delivered with nanoparticles and/or polymers described herein. Specific targeting of cells in the lymphatic system advantageously avoids systemic delivery.
DC non-activating, antigen-bearing nanoparticles are useful as a tolerogenic vaccine formulation in a number of diseases, such as type-I diabetes, for example with the antigen pro-insulin, pro-insulin-derived peptides or glutamic acid decarboxylase, among others; or such as multiple sclerosis, for example with the antigen myelin basic protein; or such as rheumatoid arthritis, for example with the antigen collagen II.
Immunotherapy with Nanoparticles and Nucleic Acids
Some nanoparticle collections may include nanoparticles and nucleic acids, e.g., DNA or RNA that encode a peptide, e.g., an antigen. Moreover, these may also include an expression cassette, include a promoter or enhancer, be part of a vector, or otherwise incorporate gene delivery motifs as are known in these arts, see, e.g., U.S. Pat. Nos. 7,160,695, 7,157,089, 7,122,354, 7,052,694, 7,026,162, 6,869,935, which are hereby incorporated by reference to the extent they do not contradict what is explicitly disclosed herein. Moreover, the sequences may encode other therapeutic biomolecules. Nucleic acids antigens such as DNA can be attached to nanoparticles, including complexation within polymer micelles or encapsulation within polymeric vesicles, or branched polymers as described for antigens and other agents herein. And, for instance, DNA-polymer conjugation can be performed through electrostatic adsorption, polymer-biotinylation [139]. A number of other chemical conjugation strategies exist to attach polymers to DNA, which may be adapted for use herein [140]. By using the nanoparticles described herein, APCs including lymph node DCs may be targeted for antigen gene expression as well as activated to ensure appropriate T cell stimulation.
Ultrasmall DNA complexes based on PEG-PPS blended with PEG-PPS-PEI may be used to deliver nucleic acids, e.g., as DNA vaccines. Targeting T cells is also useful. FIG. 32, see Example 25, shows that these ultrasmall complexes efficiently transfect cells in vivo with a model protein (green fluorescent protein, GFP). Until now, DNA vaccines have been used very nonspecifically in terms of their cellular target; herein a different approach is demonstrated, namely transfecting cells resident in the lymph node. The PEG-PPS and PEG-PPS-PEI platform provides for multifaceted approaches in vaccination. For example, hydrophobic immunomodulators, e.g., imiquimod, may be incorporated into the DNA complex. Alternatively, CpG DNA can be incorporated, either within the plasmid itself or separately within the complex. The complex can be designed to activate complement, or can be coupled to other danger signals such as flagellin. It is also useful to combine peptide/protein formulations with DNA formulations, so as to achieve a short-term presence of antigen followed by a prolonged presence due to gene expression.
DNA vaccines have been explored by others, with approaches ranging from naked DNA or complexes with cationic polymers and cationic lipids, to viral forms as well. Targeting cells within the lymph node with nonviral vectors for gene delivery, for DNA vaccines, is severely complicated by, among other factors, the size of nonviral complexes of cationic lipids or cationic polymers. With plasmid DNA, for example, these complexes are typically in the size range of 100-250 nm, and in exceptional and idiosyncratic formulations no smaller than 60-100 nm. As shown herein, however, particle trafficking via interstitial flow is very effective for very small nanoparticles, for example as shown with 20-25 nm nanoparticles herein. Example 25 demonstrates that, for the first time, ultrasmall plasmid DNA complexes can be formed that reach the lymph nodes and target the intended cell types there. These complexes were made from mixed micelles of PEG-PPS and of PEG-PPS-PEI. The PEG-PPS-PEI complexes the DNA, and the ratio of PEG-PPS to PEG-PPS-PEI controls the size of the complex. As shown in Example 25, these ultrasmall plasmid DNA complexes are capable of reaching the lymph nodes when injected, for example intradermally or subcutaneously. Transfection of T cells, B cells, and DCs was observed. Thus, the complexes taught herein are useful as DNA vaccines targeting a number of cell types. Alternatively, the complexes are generally useful for delivering imaging agents or therapeutic agents. Applications include imaging and diagnostics.
Conjugation of Moieties to Nanoparticles
A variety of schemes for attaching antigens or other moieties to nucleophilic or electrophilic functional groups are available. In general, such schemes can be adapted to the attachment of drugs or danger signals, as appropriate.
By way of example, the attachment of antigens to PLURONIC on the sold-core PPS nanoparticles of the examples is provided. Antigen conjugation to PPS nanoparticles can be accomplished by functionalizing PLURONIC (a block co-polymer of PEG and PPG) surface with proteins or peptides. Pluronic F127 (Sigma), divinylsulfone (Fluka), sodium hydride (Aldrich), toluene (VWR), acetic acid (Fluka), diethylether (Fisher), dichloromethane (Fisher) and Celite (Macherey Nagel) were used as received. The reaction was carried under argon (Messer). 1H NMR was measured in deuterated chloroform (Armar) and chemical shifts (δ) are given in ppm relative to internal standard tetramethylsilane (Armar) signal at 0.0 ppm. A solution of 15 g (1.18 mmol) of Pluronic F-127 in 400 ml toluene was dried by azeotropic distillation for 4 h using a Dean-Stark trap. The solution was cooled in an ice bath and 0.283 g (11.8 mmol) sodium hydride was added. The reaction mixture was stirred for 15 min and 3.55 ml (35.4 mmol) divinyl sulfone (Sigma-Aldrich) was added quickly. After stirring in the dark for 5 days at room temperature the reaction was quenched by adding 1.35 ml (23.6 mmol) acetic acid. After filtering over celite and concentrating the filtrate under reduced pressure to a small volume the product was precipitated in 1 liter of ice-cold diethylether. The solid was filtered off, dissolved in minimum amount of dichloromethane and precipitated in ice-cold diethylether four times in total. The polymer was dried under vacuum to yield 6.0 g and stored under argon at −20° C. prior to OVA conjugation. NMR showed the presence of vinyl sulfone and the degree of functionalization was 88%. δ=1.1 (m, CH3, PPG), 3.4 (m, CH, PPG), 3.5 (m, CH2, PPG), 3.6 (PEG), 6.1 (d, CHcis═CH—SO2) and 6.4 (d, CHtrans═CH—SO2), 6.85 (dd, CH2═CHSO2—).
Peptide or protein antigens can then be conjugated to PLURONIC vinylsulfone (VS). A model protein for investigating DC antigen presentation is ovalbumin (OVA). OVA possesses the antigenic peptides OVA257-264 and OVA323-339 which are processed by DC through MHCI and II pathways, respectively. OVA257-264 and OVA323-339 are conjugated to cysteine residues through the use of a peptide synthesizer. The peptides are then conjugated to PLURONIC-VS by reaction with its cysteine thiols. 18 mg of peptide is solubilized in 6.43 ml 0.1 M sodium phosphate buffer at pH 8.5 to yield a 2 mM solution. 60 mg (1.68 mM) PLURONIC-VS are added. The mixture is stirred during 3 h and aliquots for thiol detection by Ellman's are taken every 15-30 min. 4 mg of Ellman's reagent (5,5′dithio-bis-(2-nitrobenzoic acid)) are solubilized in 1 ml of 0.1 M sodium phosphate and 1 mM EDTA (to chelate divalent metal ions, which can oxidize sulfhydryls) at pH 8.5. 15 μl is mixed with 1.5 ml 0.1 M sodium phosphate and 30 μl of the reaction mixture quenched with 120 μl of sodium phosphate at pH 7. Following mixing and incubating at room temperature for 15 min, the absorbance is measured at 412 nm. The quantity of thiols is calculated as: c=Abs/E*D, with E (Extinction factor)=14150 M−1 and D being a dilution factor. The PLURONIC was dialyzed against water through a membrane of 6-8 kDa MWCO and the solution is freeze dried. The yield is  ̃88% with full conversion of the vinyl sulfone groups as confirmed to 1H-NMR. Conjugation of OVA to PLURONIC-VS is performed by a similar strategy by utilizing free cysteine thiols and lysine amines [109-111]. The PL-VS-OVA peptide/protein is then stored at −20° C. until used for nanoparticle synthesis.
Antigen may be conjugated to the nanoparticles by other means including covalent conjugation to amino acids that are exogenous to the naturally occurring polypeptide antigen, covalent conjugation to amino acids that are endogenous to the naturally occurring antigen, physiochemical adsorption, and other means.
Embodiments include nanoparticles with one or more free nucleophilic groups available for further reaction. Nucleophilic groups include, for example, hydroxyls, amines, and thiols. Nanoparticles with carboxyl groups for subsequent reaction may also be made. FIG. 24A describes a synthetic route for preparing nanoparticles that have amines available for reaction with moieties. FIG. 24B describes nanoparticles with hydroxyls available for further reaction. FIG. 24C shows a scheme for reacting a hydroxyl or amine to react with other moieties. Many schemes are available for reacting with hydroxyls, amines, and thiols, e.g., as in Bioconjugate Techniques, 2nd Edition by Greg T. Hermanson, published by Academic Press, Inc., 2008, 1202 pages, which is hereby incorporated by reference herein. Examples include conjugation using N-hydroxysuccinimide esters, aldehydes, isocyanates, tresyl chlorides, or hydrazine/hydrazides. FIG. 25 depicts a scheme using disulfide bonds to accomplish attachment of a moiety to a nanoparticle. FIG. 26 shows that the peptide moiety SIINFEKL (SEQ ID NO:2) was successfully immobilized to nanoparticles and delivered to the lymph node, followed by uptake to lymph node-resident DCs. Here, the disulfide link is particularly useful, in that it is reduced after cellular internalization to liberate the free peptide, allowing for presentation on MHC I or MHC II. If the peptide or protein antigen contains a site that is sensitive to the proteases involved in antigen processing, either an endogenous site or an engineered site, such a scheme may be helpful but not necessary. If a peptide is utilized without such a site, it may be convenient to attach the peptide to the nanoparticle via a reducible linkage, as taught above and in FIG. 26. While conjugation via a number of sites may be contemplated and immunological responses determined to find an optimal site of conjugation, conjugation via an N-terminally located or near N-terminally located cysteine residue is often convenient, because the fidelity of fit between the antigenic peptide and its cognate receptor is higher, and thus more discriminating, than at the N-terminus. FIG. 27 shows a scheme for reacting N-terminal alpha amines of a peptide (a term that includes proteins) or other moiety to a nanoparticles, e.g., with a pyridyl disulfide or a vinyl sulfone moiety; NP represent a nanoparticle and “Flic” represents a peptide.
In addition to covalent grafting of moieties (e.g., antigens, peptides, nucleic acids) on the nanoparticle or branched polymers, physicochemical means are also available. One powerful method is synthesis or expression of a peptide or protein antigen to incorporate a strongly anionic stretch of amino acids, such as an oligoE or oligoD region, or other anionic amino acids, or a combination of anionic amino acids to enable formation of an electrostatic complex, as for siRNA or aptamer complexes. For instance, a peptide that is desired for association with a nanoparticle may be made with a plurality of aspartic acid or glutamic acid residues, e.g., 3-30; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., at least 4 or between 5 and 15. Another method that may be used is to incorporate the moiety, e.g., an antigen peptide or protein, within watery-core vesicles, for example formed with PEG-ss-PPS. Polymer vesicles may be formed from amphiphilic copolymers, as taught in Velluto et al., Molecular Pharmaceutics 2008, “PEG-b-PPS Diblock Copolymer Aggregates for Hydrophobic Drug Solubilization and Release: Cyclosporin A as an Example”, which is hereby incorporated herein by reference. Encapsulation in polymer vesicles from with a reduction-sensitive linkage within them are particularly powerful in targeting release within the cell, in that the disulfide is reduced after endocytosis, as taught in Cerritelli et al., Biomacromolecules 2007:1966-1972 which is hereby incorporated herein by reference. Accordingly, reduction-sensitive linkages may be incorporated into a micelle or nanoparticle, e.g., by forming a nanoparticle that degrades upon reaction of its disulfide bridges in situ after exposure to a physiological environment in a patient.
FIG. 34, demonstrates release after cellular uptake as a very effective delivery of a water-soluble payload to the cytoplasm of DCs, as demonstrated by delivery of a fluorescent reporter to bone marrow-derived DCs, see FIGS. 34A and 34B. Since antigen delivery to the cytoplasm is particularly important in antigen presentation by the MHC I pathway, antigen encapsulated within the watery core of the polymeric vesicles is a particularly powerful means of antigen delivery, not requiring chemical conjugation of the antigen at all. Additionally, other agents such as danger signals may be conjugated to the surface of the polymer vesicle, or incorporated within the watery core of the polymeric vesicle, or encapsulated within the hydrophobic lamella that forms the shell of the vesicle; additionally, the polymer vesicle surface can be designed to activate complement, as taught with other polymer nanoparticles, including micelles, herein.
Immunotherapy with Nanoparticles and Danger Signals
Another immunotherapy approach involves the application of danger signals. The results herein show that hydroxylated and/or thiolated nanoparticles or branched polymers may be made to provide the function of a danger signal or stimulus that matures DCs as evidenced by, e.g., the increase in the expression of the co-stimulatory molecules CD86 and CD80. While the nanoparticles or branched polymers do not require a non-complement danger signal or maturation signal, in some cases adding such a signal may further assist in the development of an immunological response. Other DC targeting studies have suggested that after antigen uptake, danger signals such as inflammatory cytokines (i.e., CD40 ligand) and/or activators of the Toll-like receptors (e.g., LPS and CpG DNA) are necessary to mature DCs and subsequently induce cell-mediated immunity [113-115]. Danger signals are identified as biomolecules that lead to upregulation of the gene NF-κB that in turn leads to maturation of APCs and release of inflammatory cytokines. In such a case a danger signal cytokines such as CD40 ligand, GM-CSF or a Toll-like receptor activator could be attached to a nanoparticle (e.g., at the core or surface layer or hydrophilic polymer layer), e.g., following previously described protocols for protein or peptide conjugation [109-111]. Moreover, a nanoparticle can be synthesized with an antigen and/or a non-complement danger signal attached to its surface.
Embodiments include nanoparticles and/or branched polymers associated with a danger signal. The danger signal may be covalently attached or adsorbed, e.g., as described elsewhere herein. The nanoparticle and/or branched polymer may have thiols and/or hydroxyls in combination with the danger signal or some or all of its available termini or surface groups derivatized with the signal. Example 22 and FIG. 21 describe experiments with nanoparticles loaded with imiquimod, a chemical danger signal currently used as a drug, were highly effective and comparable to LPS. FIG. 29 (see Example 23) provides an example of a danger signal covalently coupled for delivery wherein nanoparticles were reacted with CpG DNA by way of example. Danger signals include compounds currently investigated as adjuvant molecules, such as flagellin, flagellin fragments, CpG oligonucleotides, imiquimods, monophosphoryl lipid A, and saponins, among others.
Immunotherapy for Antibody Production
Some embodiments are directed to introducing an antigen to a patient to generate antibodies in the patient against the antigen. For example, vaccinations or antitumor therapies may be pursued in this manner. Alternatively, such approaches may be used to generate antibodies for use as scientific reagents, e.g., in animals.
Thus a combination of nanoparticle and antigen may be introduced into the patient. After a predetermined time (e.g., 1-30 days), a sample is taken from the patient and the antibodies against the antigen are measured. Additional samples and measurements may be periodically taken. If antibody titers are too low, the nanoparticles and antigens may be reintroduced and additional measurements made, with the process being repeated as necessary to bring antibody titers to a desired level. The combination may be administered several times.
Discussion of Experimental Results
Delivery of biodegradable nanoparticles to antigen-presenting cells (APCs), specifically dendritic cells (DCs) and additionally macrophages as well as B cells is documented herein, including applications for immunotherapy. Detailed examples herein describe the delivery of 20, 25, 45, and 100 nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. The nanoparticles in the detailed examples comprise a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades to soluble polymer within oxidative environments. Peptide or protein antigens, including glycopeptide (defined herein as glycosylated polypeptides) antigens, and nucleic acid encoding antigens can be attached to the nanoparticle surface. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45 nm particles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120 h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of an exogenous targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. An increase in nanoparticle-containing DCs (and other APCs) was seen at 96 h vs. 24 h, showing an infiltration of these cells to lymph nodes. Both nanoparticle size and surface chemistry were found to influence DC maturation after in vivo injection.
The basic PPS nanoparticles as synthesized, i.e., PPS nanoparticle cores stabilized with PLURONIC (a block copolymer of polyethylene glycol (PEG) with polypropylene glycol), were found to activate DCs after in vivo injection of the nanoparticles, as indicated by increased expression of the maturation markers CD86, CD80 and CD40, when the nanoparticles were very small; 25 nm nanoparticles activated DCs extensively after in vivo injection, while 100 nm nanoparticles did not. When a second nanoparticle surface chemistry was utilized, i.e., PPS nanoparticle cores stabilized with a methoxy-terminated PLURONIC, even the very small nanoparticles did not activate DCs after in vivo injection. The PLURONIC-stabilized nanoparticles were demonstrated to effectively activate complement whereas the nanoparticles stabilized with methoxy-terminated PLURONIC were not effective to activate complement. Selected formulations bearing surface thiols where also found to induce complement activation, and data presented herein indicates that thiol-activated complement may be particularly powerful in triggering an adaptive immune response. Thus, nanoparticle complement activation induced DC activation after exposure to these nanoparticles.
Both nanoparticle size and surface chemistry were found to influence adaptive immunity after in vivo injection. Antigen was conjugated to PLURONIC-stabilized nanoparticle surfaces and was found to strongly induce antibody formation only when the nanoparticles were very small; 25 nm nanoparticles induced antibody formation much more strongly than 100 nm nanoparticles. When a second nanoparticle surface chemistry was utilized, i.e., PPS nanoparticle cores stabilized with a methoxy-terminated PLURONIC, even the very small nanoparticles did not induce strong antibody formation after in vivo injection. Moreover, when 25 nm PLURONIC-stabilized nanoparticles were injected in mice in which complement protein 3 had been knocked out (C3−/− mice), these nanoparticles did not strongly induce antibody formation. Thus, the detailed examples show that nanoparticles of suitable size, e.g., of 20-45 nm, have the potential for immunotherapeutic applications; for example, they may be used to specifically target and activate DCs in lymph nodes. Moreover, when these nanoparticles possess a surface chemistry that activates complement, such as obtained by stabilization by PLURONIC, they have strong potential for function as an antigen carrier and adaptive immunity-inducing adjuvant. The special combination of small size (e.g., 20-45 nm) and complement activation is valuable in vaccine formulations as an adjuvant.
The examples thus show that nanoparticles can be used for targeted antigen and drug delivery to APCs, specifically DCs, in lymph nodes. The simplicity of this approach is that by controlling size, nanoparticles can be effectively taken up into lymphatics as well as retained in lymph nodes (as shown, for at least 5 d), and without using any specific exogenous targeting ligand; they are internalized effectively by nodal resident DCs and other APCs (e.g., macrophages). Nanoparticles as large as about 45 nm or up to about 100 nm can not be effectively targeted into the lymphatics and the lymph node by this means. Additionally, it is demonstrated that up to about 40 to about 50% of resident lymph node DCs internalize nanoparticles, further demonstrating the effectiveness of this delivery vehicle. Also it was demonstrated that following exposure to nanoparticles of such size ranges as to be effectively targeted to the lymphatics, when those nanoparticles activate complement, the DCs respond by becoming more mature and inducing T cell dependant adaptive immunity. This is a clear demonstration of the potency of using the complement cascade as a danger signal in antigen presentation adjuvant formulations. It was demonstrated that the combination of small size (so as to effectively enter the lymphatics after administration) and complement activation (so as to stimulate APC including DC maturation) that strong adaptive immune responses could be induced, both T cell-dependant humoral (via Ab titers) and cellular (via the measurements of T-cell memory via T-cell proliferation and ELISPOT measurements). This special combination of small size and complement activation is very valuable in immunotherapeutics. Experiments presented herein demonstrate that the TLR4 receptor may play a role in this response as well. Activation of the adaptive immune response via complement may be very valuable from the perspective of safety and side-effects, in that production of potent, and dangerous, inflammatory cytokines such as IL-6 and TNFalpha were very low from DCs stimulated with complement-activating nanoparticles compared to those stimulated with LPS or with CpG. Accordingly, embodiments include an immunostimulatory composition for introduction into a patient that comprises a collection of synthetic nanoparticles that comprise an antigen, with the composition inducing an immune response against the antigen in the patent without inducing a TNF-alpha response and without inducing an IL-6 response.
Other nanoparticle forms are demonstrated in the Examples. Micelles formed from amphiphilic block copolymers are presented, using block copolymers of PEG and PPS and of PEG, PPS and PEI as an experimental example. Micelles are very convenient, in that they can be formed to very small size and with very narrow size distribution. Their surfaces can be designed to activate complement via surface hydroxyls or surface thiols. Hydrophobic danger signals or immunosuppressants can be incorporated within the micelle core. Antigen can be chemically conjugated or complexed to the micelle surface. The Examples demonstrate formation of micelles, conjugation with antigen, activation of complement, activation of DCs, and induction of adaptive immunity. The Examples demonstrate formation of very small nanoparticles of plasmid DNA with polymer nanoparticles and efficient transfection of DCs, B cells and T cells within the lymph nodes of the lymphatics that drain the injection site. Thus, the Examples show the ability to use such forms from block copolymers for both peptide/protein antigens and for DNA-encoded antigens. The examples teach the utility of cells within the lymph node as a target for both peptide/protein antigens and for DNA-encoded antigens.
Other nanoparticle forms from block copolymeric amphiphiles are demonstrated in the Examples. Watery-core vesicles, sometimes referred in the literature as polymersomes or polymerosomes, are formed from block copolymers, using block copolymers of PEG and PPS as an experimental example. Vesicles are very convenient, in that they can contain a hydrophilic payload within their watery core. This hydrophilic payload may comprise hydrophilic danger signals, such as CpG oligonucleotides, or antigens, such as peptides or proteins, or gene sequences that encode the expression of antigens, other immunomodulatory proteins, or more than one of the above.
Other nanoparticle forms from branched polymers are demonstrated in the Examples. Nanoparticles can be so small, that they can be effectively formed from a single, branched polymer. Branching provides multiple sites for conjugation of antigen, or conjugation of danger signals or spontaneous assembly with danger signals such as complement, or both. The Examples present approaches by which to synthesize such functionalized branched polymers.
The Examples demonstrate the use of lymph node-targeting nanoparticles for both induction of an adaptive immune response and for induction of tolerance. As described in detail herein, and in the Examples, antigens and danger signals can be conjugated to or complexed to or encapsulated within nanoparticles, including micelles and vesicles, or branched polymers for inducing an adaptive immune response; or antigens can be conjugated to or complexed to or encapsulated within nanoparticles, including micelles and vesicles, or branched polymers in the absence of danger signals, or even along with immunosuppressant or immunomodulatory molecules for inducing tolerance. Analogously, antigen-encoding DNA can be delivered either with danger signals to induced adaptive immunity or in the absence of danger signals or even along with immunosuppressant or immunomodulatory molecules for inducing tolerance.
The Examples focus experimental data on PPS as a hydrophobic component of a polymer nanoparticles, including micelles and vesicles. PPS is stable in the presence of water, but is unstable in the oxidative conditions of the body and especially in the oxidative conditions of the lysosome after endocytosis. As such, the polymer is oxidized from the hydrophobic polypropylene sulfide to the hydrophilic sulfoxide and sulfone, resulting after oxidation of a polymer that is water soluble and can be cleared by the kidney via renal filtration. As such, PPS and its copolymers can be stable when formulated in water in a vial, but are unstable after injection in the body. This provides distinct cost and logistic advantages, which may be particularly important for applications. The nanoparticles can further be dried by lyophilization or by simple drying and can be rehydrated to re-form an injectable nanoparticulate formulation. In some applications, the dry formulation may be directly useful, for example when placed on a mucosal tissue surface.
The Examples also demonstrate different routes of administration. When injected within a tissue interstitium, such as intradermally, subcutaneously, or intramuscularly, the ultrasmall size of nanoparticles permits their permeation through the interstitium under the convective influence of interstitial flow, flowing from blood capillaries in the tissue to lymphatic capillaries or clearance into the draining lymph nodes. If the particles are sufficiently small, they are not entrapped in the interstitium and are thus effectively transported into the lymphatics. The Examples also demonstrate the application of exogenous pressure to obtain penetration across a tissue barrier, for example penetration into the oral mucosa with the application of a mild pressure to sufficiently small nanoparticles. Mild pressure is a term that excludes high pressure needeless injection systems, which rely on high pressures to force medicaments into a patient. Mild pressure advantageously avoids actual mucosal disruption. Certain embodiments are directed to use of an exogenous pressure of less than about 760 mm Hg; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., less than about 40, 100, or 200 mm Hg or from about 10 to 120 mm Hg. Examples of external mucosa are nasal, buccaly, oral, vaginal, and colonic, e.g., delivered by buccaly, pessary or suppository means.
EXAMPLES
Example 1
Nanoparticles
PPS nanoparticles with diameters of 20, 45, and 100 nm were synthesized by inverse emulsion polymerization as described elsewhere; in the term “emulsion polymerization” used herein, is included inverse emulsion polymerization, and in the term “emulsion”, is included inverse emulsion [39]. Briefly, an emulsion was created by adding the PEG block copolymer emulsifier, PLURONIC F-127 (Sigma-Aldrich, Buchs, Switzerland) and the monomer propylene sulfide to ultrapure milliQ water under constant stirring. The protected initiator pentaerythritol tetrathioester was synthesized as described elsewhere [39] and, in a separate flask, was deprotected by mixing it with 0.20 mL of 0.5 M sodium methylate solution under stirring for 10 min. Following deprotection, the initiator was then added to the monomer emulsion and 5 min later 60 μl of the base diaza[5.4.0]bicycycloundec-7-ene (DBU) was added to the reaction and it was allowed to stir continuously for 24 h under an inert atmosphere. The nanoparticles were then exposed to air in order to produce disulfide cross-linking.
The nanoparticles were purified from remaining monomers, base, or free PLURONIC by 2 d of repeated dialysis with a 12-14 kDa MWCO membrane (Spectrum Laboratories, Rancho Dominguez, Calif.) against ultrapure milliQ water. The nanoparticle size distributions were determined by the use of a dynamic light scattering instrument (Malvern, Worcestershire, United Kingdom). Fluorescent labeling was accomplished by adding 6-iodoacetamido-fluorescein or Alexa Fluor 488 maleimide (Molecular Probes, Eugene, Oreg.) at 1 mg/ml of nanoparticle solution to the reactive thiols remaining on the nanoparticles, and then stirred in the dark for 6 h. The nanoparticles were then exposed to air for further disulfide cross-linking. Free iodoacetamido-fluorescein or Alexa Fluor maleimide was eliminated by repeated dialysis for 1 d using a 25 kDa MW cutoff membrane (Spectrum Laboratories) against 5 mM PBS.
Example 2
Synthesis of Methoxy-Terminated PLURONIC




Pluronic F127 (Sigma), methyl iodide (Fluka), potassium hydroxide (Fluka), sodium thiosulfate penta hydrate (Riedel de Haen), anhydrous sodium sulfate (Applichem), sodium chloride (Sigma), diethylether (Fisher) and dichloromethane (Fisher) were used as received. Tetrahydrofuran stabilized with 0.025% BHT (Acros) was dried over molecular sieves before use. The reaction was carried out under argon (Messer) atmosphere. For dialysis a regenerated cellulose tube (Spectrapor) with molecular weight cut off 3400 was used. 1H NMR was measured in deuterated dimethylsulfoxide (Armar) and chemical shifts (δ) are given in ppm relative to residual solvent signal at 2.5 ppm.

To a solution of 10.0 g (0.79 mmol) PLURONIC F127 in 100 ml THF was added 2.99 g (53.3 mmol) finely ground potassium hydroxide and 988 μl (15.9 mmol) methyl iodide and the mixture stirred in the dark for 19 h. The clear solution is decanted and 3.94 g sodium thiosulfate penta hydrate, 100 ml saturated aqueous sodium chloride solution and 100 ml dichloromethane is added. The mixture was stirred vigorously and transferred to a separating funnel. The layers were separated and the aqueous phase extracted with dichloromethane (2×100 ml). The organic fractions were combined, dried over sodium sulfate and concentrated under reduced pressure. The solid was dissolved in a minimum amount of bidistilled water and dialyzed against 4500 ml water for one day. The clear aqueous solution is saturated with sodium chloride and extracted with dichloromethane (3×100 ml) and dried over sodium sulfate. After solvent removal the residue is extracted in a soxhlet extractor with diethylether for 6 h to yield after drying under reduced pressure 9.25 g of white solid. NMR showed the absence of the OH group at 4.6 ppm and the presence of the OCH3 group at 3.2 ppm. δ=1.1 (d, CH3, PPG), 3.2 (s, OCH3) 3.3 (m, CH, PPG), 3.4 (m, CH2, PPG), 3.5 (m, PEG).
Example 3
Animals
Unless otherwise stated, BALB/c mice, 6-9 weeks old and weighing 20-30 g, were used for this study. All protocols were approved by the Veterinary Authorities of the Canton Vaud according to Swiss law. Anesthesia was delivered by subcutaneous injection of ketamine hydrochloride at 10 mg/kg and xylazine at 1 mg/kg. Mice were euthanized by CO2 asphyxiation.
Example 4
Microlymphangiography
To determine the relative uptake characteristics of the nanoparticles following interstitial injection into the skin, fluorescence microlymphangiography was performed by constant pressure injection into the tip of the tail as previously described [43-45]. Hair was depilated from the mouse tail, and the mouse was positioned on the microscope stage (Axiovert 200M, Zeiss) with a heating pad to maintain 37° C. body temperature. A 20 mg/ml solution of fluorescent PPS nanoparticles (20, 45, or 100 nm diameter) in sterile phosphate-buffered saline (PBS) was drawn into a catheter; a 30-gauge needle attached to the catheter was inserted intradermally at  ̃1 mm from the tail tip and a stopcock was opened that initiated flow at a constant pressure of 40 mm Hg. The flow rate of the nanoparticle solution (monitored with a bubble far upstream in the tubing) averaged 0.1 μl/min into roughly 20 mm3 of tissue (approximated from the volume visible from the injection depot), or approximately 5 μl/g/min. No visible swelling was observed. Sequential images along the length of the tail were collected at a constant exposure time; 3 experiments were repeated for each nanoparticle size.
Fluorescence microlymphangiography in the mouse tail was used to evaluate the lymphatic uptake of PPS nanoparticles of 20, 45, and 100 nm diameter. Following infusion with 20 nm particles, the hexagonal lymphatic capillary network was clearly visible after 90 min and uniformly filled from the injection site (FIG. 1a). In contrast, only a very faint lymphatic network was observed following injection with the 45 nm particles (FIG. 1b), and very little network could be seen with the 100 nm particles (FIG. 1c), indicating poor uptake. The upper limit of size for macromolecule/protein/particle leakage into blood capillaries is well known to be  ̃3.5 nm [42], therefore the leakage into blood vasculature of our smallest 20 nm particles is effectively zero. This method thus qualitatively confirms that 20 nm particles are more readily taken up into the lymphatic capillaries from the interstitial space compared to 45 nm and 100 nm particles.
Example 5
Evaluation of Nanoparticle Distribution in Lymph Nodes: Lymph Node Retention of Small Nanoparticles (e.g., 20-45 nm)
To evaluate lymph node retention, 20 μl of 20 mg/ml fluorescent PPS nanoparticles (20, 45, and 100 nm diameter) were injected as a bolus into the tip of the mouse tail or front footpad through a 30 gauge needle; controls were performed with 20 μl injections of PBS. No inflammation was observed at the sites of injection. At 24, 72, 96, or 120 h, mice were sacrificed by CO2 asphyxiation. The sacral and lumbar lymph nodes, which drain the tail and leg lymphatics, brachial and axillary lymph nodes, which drain the front footpad area lymphatics were removed, flash frozen, cryosectioned into 10 μm sections, and immunostained with antibodies against mouse CD3e (Pharmingen, San Diego, Calif.), CD45R (Caltag, Burlingame, Calif.), CD68 (Serotec, Dusseldorf, Germany), Dec-205 (Serotec), and CD31 (Pharmingen) to label T cells, B cells, macrophages/DCs, DCs, and endothelial cells, respectively. Secondary detection was performed with Alexa Flour 594 nm (Molecular Probes) antibodies. Lymph node sections were imaged with fluorescence (Axiovert 200M, Zeiss) and confocal laser scanning microscopy (LSM 510 Meta, Zeiss).
Lymph node retention time of nanoparticles and liposomes has been investigated by several other researchers for purposes of studying the lymphatic system and typically has focused on time points in the range of 6-52 h post-injection [31-34, 36]. Examples reported herein described lymph node retention of nanoparticles up to 120 h, and showed that 20 nm particles were significantly present at qualitatively consistent levels in the lymph node at 24, 72, 96, 120 h following a bolus injection of 20 μl intradermally (FIG. 2). The 45 nm nanoparticles were also present, although in lower amounts, in lymph nodes at all time points, while the 100 nm nanoparticles were not visibly present in the lymph nodes at any time point (FIG. 2). Thus, together with the results in FIG. 1, these data show that 20-45 nm is a good PPS nanoparticle size range for both lymphatic uptake and lymph node retention, with 20 nm being optimal, while 100 nm particles are too large for efficient lymphatic uptake from the interstitium following a constant pressure injection. This is consistent with previous studies that have shown liposomes>70 nm remain mostly at the site of injection [24, 30].
The specific locations, with respect to the various immune cells, in which PPS nanoparticles accumulated within lymph nodes, were evaluated. Staining results were consistent with known lymph node architecture, where specific zones for T and B lymphocytes can be readily seen [2]. T cells tend to aggregate in the center regions of the node while B cells are often found in germinal centers located towards the outer membrane. The other major cell types present in lymph nodes are APCs or MHCII+ cells, namely DCs and some macrophages, and their location is often more dispersed. FIG. 3 shows serial sections of the same lymph node following an intradermal injection of 20 nm particles. The nanoparticles were not present in the T cell or B cell zones (FIGS. 3a, b). However, there was significant co-localization of the nanoparticles with macrophages and DCs; i.e., CD68+ cells (FIG. 3c).
It has generally been assumed that liposomes and nanoparticles delivered to lymph nodes are primarily phagocytosed by macrophages there [21, 27, 29, 30, 32, 36, 37]. However, it has not been appreciated in these arts that immature DCs capable of taking up antigens are also present in the lymph nodes [15, 16]. With PPS nanoparticle delivery, immunostaining for CD68 (which, although a transmembrane protein, is also expressed intracellulary [47-50]) verified that macrophages and DCs had internalized PPS nanoparticles (FIG. 4a). To further determine whether the CD68+ cells were macrophages, DCs, or both, lymph nodes were immunostained for the highly-specific DC receptor Dec-205 [4, 38, 51-56]. The Dec-205+ cells and their co-localization with nanoparticles (FIG. 4b) demonstrate that a significant fraction of the cells in the lymph node that phagocytose the nanoparticles were, indeed, DCs. This will be advantageous for delivering antigens to lymph nodes in order to stimulate APCs, including the most potent APC type—DCs.
Example 6
Lymph Node Cell Isolation and Staining
Lymph node cells were isolated following a previously described protocol [46]. Briefly, following injections of fluorescent nanoparticles or PBS as described earlier, lymph nodes were removed, teased with 26 gauge needles and digested in Collagenase D (Roche, Basel, Switzerland) for 25 minutes at 37° C. Tissue was then passed through a 70-μm cell strainer (BD, Basel, Switzerland) to recover a cell suspension. With the lymph node cell suspension, APCs were stained for with anti-MHC Class II-(I-A)-R-PE (Chemicon, Temecula, Calif.) and DCs with anti-CD11c-allophycocyanin (Pharmingen). DC maturation was measured by staining with anti-CD86-R-PE and anti-CD80-R-PE (Pharmingen).
Example 7
In Vitro Nanoparticle Internalization
Following lymph node cell isolation, cells were plated in RPMI (5% FBS) at  ̃500,000 cells/ml. Cells were then pulsed with 20 μl of 20 mg/ml of fluorescent nanoparticles and incubated for 24 h. Cells were then washed twice with HBSS and stained for APCs and DCs as mentioned earlier.
Example 8
Flow Cytometry & Analysis and In Vitro Nanoparticle Internalization: Uptake by APCs, Including DCs
Following staining, lymph node cell suspensions were analyzed by flow cytometry (CyAn ADP, Dako, Glostrup, Denmark). Further analysis was performed using FlowJo software (TreeStar, Ashland, Oreg.). APCs and DCs that had internalized fluorescent nanoparticles were determined to be MHCII+FITC+ and CD11c+FITC+, respectively, FITC representing labeling of the nanoparticles. DC maturation following nanoparticle internalization was evaluated by calculating the fraction of cells that expressed CD86 and CD80.
Flow cytometry analysis was performed to quantify the fraction of APCs and DCs in lymph nodes that were internalizing nanoparticles. FIG. 5a shows that up to  ̃40% of APCs (MHCII+) and specifically  ̃50% of DCs (CD11c+) in lymph nodes have taken up 20 nm nanoparticles after 24 h post-injection. Accordingly, and in general, nanoparticle applications with at least at 10% to about 95% uptake by APCs and/or DCs are contemplated; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., at least about 25%, at least about 40%, or between about 25% and about 75%/50%. Also a significant fraction of APCs and DCs phagocytose 45 nm nanoparticles while very little uptake of 100 nm nanoparticles was observed after in vivo injection. APCs, including the DCs, could have either endocytosed the particles after they arrived in the lymph node, or internalized them at the injection site before trafficking to lymph nodes. If the latter were the case, 100 nm particles would be seen in the lymph node, since larger particles (1-10 μm) can be endocytosed by APCs just as efficiently as smaller ones [13, 57]. It was verified that nanoparticle size did not affect APC or DC internalization by in vitro experiments; nearly all APCs and DCs internalized nanoparticles regardless of size (FIG. 5b). Therefore PPS nanoparticles are likely taken up passively into peripheral lymphatic vessels and reach the lymph nodes, where they are phagocytosed by resident DCs or APCs. These results reinforce the recent findings that substantial numbers of immature DCs capable of internalizing antigens are present in lymph nodes [15, 16]. Indeed, DCs and other APCs in lymph nodes offer a valuable target for initiating cell-mediated immunity via drug delivery vehicles.
A comparison of nanoparticle internalization at different times was investigated. It was found that nanoparticle co-localization with macrophages and DCs was visibly higher at 96 h than at 24 h (FIG. 6a). Flow cytometry analysis was used to determine if there was a change in the type of macrophages and DCs internalizing nanoparticles at 96 h vs. 24 h. Of all cells that had internalized nanoparticles at 24 h,  ̃15% were APCs (MHCII+) and  ̃13% were DCs (CD11c+), suggesting that most of the APCs were DCs, and that the remaining  ̃85% of nanoparticle containing cells were non-antigen presenting macrophages (MHCII−). At 96 h, the fraction of cells with nanoparticles that were APCs or DCs was 61±5% and 33±3%, respectively (Note that this does not reflect an increase in the fraction of lymph node APCs and DCs that contain nanoparticles, which remains constant at levels shown in FIG. 5a). The increase in nanoparticle-containing MHCII+ and CD11c+ cells may be due to an infiltration of APCs and DCs into lymph nodes that then pick up free nanoparticles still remaining in the nodal tissue. It is also possible that the increase in MHCII+ cells with nanoparticles is due to the activation of macrophages between 24-96 h (i.e., MHCII− macrophages that become activated and thus MHCII+ following nanoparticle internalization).
A significant fraction of DCs in lymph nodes are already mature, however since there are immature DCs present, it is likely that these cells become mature following antigen uptake. Therefore it was determined if 20 nm PLURONIC-stabilized complement-activating (and thus hydroxylated) PPS nanoparticles help induce DC maturation such that conventional biological exogenous “danger signals” were not necessary when used in combination with the PLURONIC-stabilized complement-activating PPS nanoparticles. Following injections of 20 nm PLURONIC-stabilized complement-activating nanoparticles, the expression of the DC maturation marker CD86 was increased compared to controls that received injections of PBS (FIG. 7). The DC expression of CD80 was also measured and determined to be significantly greater following nanoparticle internalization (FIG. 7b). Finally it was found that the expression levels of CD86 and CD80 in DCs with nanoparticles did not change at 96 h vs 24 h post-injection. This suggests that the 20 nm PLURONIC-stabilized complement-activating nanoparticles offer a prolonged maturation stimulus, which could be useful for maintaining T cell activation and cell-mediated immunity over a prolonged time period. Therefore these results show that PLURONIC-stabilized complement-activating PPS nanoparticles may serve a dual role, acting as a vehicle for DC specific antigen delivery and also as an adjuvant that matures and activates DCs in lymph nodes.
Example 9
C3a Detection in Human Serum
96 well plates (Becton Dickinson) were coated with C3a/C3a (desArg) mouse anti-human monoclonal antibody (AntibodyShop, Grusbakken, Denmark) at 1:4000 dilution in PBS at 100 μl/well. Plates were left overnight at room temperature (RT). Unbound antibody was flicked off (i.e., removed by sudden mechanical motion) and the plate was washed 3× with 200 μl/well DI water. Plates were then blocked for 1.5 h at RT with 200 μl/well blocking buffer (PBS+Tween20 at 0.05%+Bovine serum albumin at 0.5%).
Human serum was incubated at 1:1 volume with PBS, suspensions of nanoparticles stabilized with PLURONIC (and thus hydroxylated nanoparticles), and suspensions of nanoparticles stabilized with methoxy-terminated nanoparticles (and thus not hydroxylated nanoparticles) in Eppendorf tubes at 37° C. for 45 min. When plates were finished blocking, they were washed 3× with 250 μl/well wash buffer (PBS+Tween20 at 0.05%). Serum-nanoparticle samples were then added to wells in triplicate at a 50 μl/well for 2 h at RT. Samples were then flicked out and plate is then washed with wash buffer 5× at 250 μl/well. C3a/C3a (desArg)-biotinylated detection antibody (AntibodyShop) was then added at 1:4000 dilution in blocking buffer at 50 μl/well for 2 h at RT. C3a detection antibody was then flicked out and the plate was washed with wash buffer 5× at 250 μl/well. Next streptavidin-HRP antibody (R&D systems) was diluted in blocking buffer at concentration recommended by manufacturer and added to plate at 50 μl/well for 2 h at RT. HRP antibody was then flicked out and the plate was washed with wash buffer 5× at 250 μl/well. Next, HRP substrate reagent (R&D systems) was added at 100 μl/well in the dark for 45 min at RT. The reaction was stopped by adding 50 μl/well of 2N H2SO4. The plate was then read on Tecan plate reader at 450 nm and 540 nm wavelength. The 540 nm background values were subtracted from 450 nm to obtain final values.
The C3a present in serum incubated with PBS was compared to C3a present following incubation with PPS nanoparticles stabilized with PLURONIC (and thus hydroxylated nanoparticles) and PPS nanoparticles stabilized with methoxy-terminated PLURONIC (and thus not hydroxylated nanoparticles). Incubation with the PPS nanoparticles stabilized with methoxy-terminated PLURONIC lead to  ̃7 fold increase, and incubation with the PPS nanoparticles stabilized with PLURONIC lead to a  ̃32 fold increase of C3a present in serum with PBS, as shown in FIG. 8. The hydroxylated nanoparticles thus activated the complement system much more than non-hydroxylated nanoparticles as measured by the cleavage of human serum C3 into soluble C3a and bound C3b. This confirms that the nanoparticle's OH surface is activating the alternative pathway of the complement system far more efficiently than a methoxy surface.
Example 10
Surface Chemistry Effects on DC Maturation
25 nm PLURONIC-stabilized nanoparticles (and thus hydroxylated nanoparticles; produced as described herein), 25 nm nanoparticles stabilized with methoxy-terminated PLURONIC (and thus not hydroxylated) nanoparticles, 20 nm carboxylated polystyrene nanospheres (COOH—NSs) (Invitrogen), PBS, and LPS (30 μg) were injected intradermally into mice as previously described. Lymph nodes were then harvested and cells are isolated and stained for CD11c, CD86, CD80, and CD40 as previously described. Flow cytometry was performed to determine maturation profile of lymph node DCs.
As seen in FIG. 9, the CD86, CD80, and CD40 profiles for PLURONIC-stabilized (and thus hydroxylated) nanoparticles compared to methoxy-terminated PLURONIC-stabilized nanoparticles (and thus not hydroxylated) nanoparticles and to carboxylated polystyrene nanospheres are significantly different. The DCs from animals treated with PLURONIC-stabilized nanoparticles show higher expression of these DC maturation markers, furthermore they induce maturation to similar levels as the positive control LPS. The DCs from animals treated with methoxy-terminated PLURONIC-stabilized nanoparticles and from animals treated with carboxylated polystyrene nanospheres yielded nearly identical DC maturation responses as negative control PBS injection. These results show that the DC maturation response in vivo is specifically dictated by the surface chemistry of 20-25 nm nanoparticles. The hydroxylated surface induces DC maturation while the methoxy and carboxyl surfaces do not. Based on the results presented herein, the functional difference in these surfaces is in complement activation by the hydroxylated surfaces.
Example 11
Size Effects on DC Maturation
25 nm PLURONIC-stabilized (and thus hydroxylated) nanoparticles, and 100 nm PLURONIC-stabilized (and thus hydroxylated) nanoparticles, and PBS were injected intradermally into mice as previously described. Lymph nodes were then harvested and cells were isolated and stained for CD11c, CD86, CD80, and CD40 as described herein. Flow cytometry was performed to determine maturation profile of lymph node DCs.
As seen in FIG. 10, the CD86, CD80, and CD40 profiles for 25 nm PLURONIC-stabilized (and thus hydroxylated) nanoparticles compared to 100 nm of the same surface chemistry are significantly different. The 25 nm PLURONIC-stabilized nanoparticles show higher expression of these DC maturation markers. The 100 nm PLURONIC-stabilized nanoparticle injections yielded nearly identical DC maturation responses as negative control PBS injection. These results show that the DC maturation response in vivo is specifically related to nanoparticle size. It has been described herein that 25 nm PLURONIC-stabilized nanoparticles efficiently enter lymphatic capillaries and traffic to lymph nodes to much greater extent than 100 nm PLURONIC-stabilized nanoparticles. Also the retention of 25 nm PLURONIC-stabilized nanoparticles and internalization by DCs in lymph nodes is much greater than that of 100 nm PLURONIC-stabilized nanoparticles. Here it is shown that also the DC maturation is much greater with 25 nm PLURONIC-stabilized nanoparticles compared to 100 nm PLURONIC-stabilized nanoparticles. The ability of ultrasmall 25 nm PLURONIC-stabilized nanoparticles to induce DC maturation demonstrates that lymph node targeting and surface chemistry are useful to activate DCs. Based upon the results on entry of nanoparticles into the lymphatic capillaries presented herein, it is expected that 45 nm PLURONIC-stabilized (and thus hydroxylated) nanoparticles can activate DCs within the lymph node.
Example 12
OVA Conjugation to Nanoparticles
Antigen conjugation to PPS nanoparticles can be accomplished by functionalizing Pluronic (a block co-polymer of PEG and PPG) surface with proteins or peptides, including glycopeptides. A conjugation scheme is presented in this example for a protein antigen that contains a free cysteine residue for chemical conjugation. Other functionalities can be used in related schemes, such as amines at the N-terminus or on lysine residues. Antigen may also be adsorbed to nanoparticle surfaces.
Here, shown is the conjugation scheme for ovalbumin (OVA). OVA is a model protein for investigating DC antigen presentation that possesses the antigenic polypeptides OVA257-264 and OVA323-339 which are processed by DC through MHCI and II pathways, respectively. The conjugation scheme starts with synthesis of Pluronic divinylsulfone to which OVA is coupled via a free thiol group on OVA in a Michael addition reaction. Synthetic details for both steps are given below.
Pluronic F127 (Sigma), divinylsulfone (Fluka), sodium hydride (Aldrich), toluene (VWR), acetic acid (Fluka), diethylether (Fisher), dichloromethane (Fisher) and Celite (Macherey Nagel) were used as received. The reaction was carried under argon (Messer). 1H NMR was measured in deuterated chloroform (Armar) and chemical shifts (δ) are given in ppm relative to internal standard tetramethylsilane (Armar) signal at 0.0 ppm.
A solution of 15 g (1.18 mmol) of Pluronic F-127 in 400 ml toluene was dried by azeotropic distillation for 4 h using a Dean-Stark trap. The solution was cooled in an ice bath and 0.283 g (11.8 mmol) sodium hydride was added. The reaction mixture was stirred for 15 min and 3.55 ml (35.4 mmol) divinyl sulfone (Sigma-Aldrich) was added quickly. After stirring in the dark for 5 days at room temperature the reaction was quenched by adding 1.35 ml (23.6 mmol) acetic acid. After filtering over celite and concentrating the filtrate under reduced pressure to a small volume the product was precipitated in 1 liter of ice-cold diethylether. The solid was filtered off, dissolved in minimum amount of dichloromethane and precipitated in ice-cold diethylether four times in total. The polymer was dried under vacuum to yield 6.0 g and stored under argon at −20° C. prior to OVA conjugation. NMR showed the presence of vinyl sulfone and the degree of functionalization was 88%. δ=1.1 (m, CH3, PPG), 3.4 (m, CH, PPG), 3.5 (m, CH2, PPG), 3.6 (PEG), 6.1 (d, CHcis═CH—SO2) and 6.4 (d, CHtrans═CH—SO2), 6.85 (dd, CH2═CHSO2—).
Prior to conjugation PLURONIC vinylsulfone was dialyzed against water using a regenerated cellulose dialysis tube with molecular weight cut off of 6-8 for kDa days. The material was recovered by lyophilization and NMR is measured to confirm that this step has no influence on the number of vinyl sulfone groups. Conjugation of OVA is done by adding 300 mg (0.023 mmol) PLURONIC vinylsulfone to a solution of 50 mg (0.0011 mmol) OVA in 0.1 M sodium phosphate buffer (pH=8.1). After reacting for 6 h at 4° C. the reaction mixture was lyophilized. Dichloromethane is added and the turbid mixture centrifuged at 12000 rpm for 5 min at room temperature. The dichloromethane, containing unreacted PLURONIC vinylsulfone, is removed and the precipitate dried under reduced pressure to remove residual dichloromethane. The PL-VS-OVA was then stored at −20° C. until used for nanoparticle synthesis.
Particles were synthesized as described herein with the difference that 2% wt of the Pluronic was replaced with PL-VS-OVA. A total amount of 1.5% PLURONIC to PPS (weight/volume) was used. In order to reduce the exposure of the protein or peptides to the basic conditions during nanoparticle synthesis reaction time was reduced to 6 h and base was added at 1:1 molar ratio to initiator-thiols.
In addition PL-VS-OVA can be fluorescently labeled with rhodamine iodoacetamide by reacting the remaining free thiols on OVA. Nanoparticles can be synthesized and labeled with flourescein iodoacetamide to produce dual labeled OVA-conjugated nanoparticles, where OVA is labeled with rhodamine and nanoparticles with fluorescein.
Dual labeled OVA-conjugated nanoparticles were injected intradermally into mice as described herein. Lymph nodes were then removed at 24 and 48 h post-injection, they were then frozen and cryosectioned. The lymph node sections were then viewed by fluorescence microscopy.
Dynamic light scattering was performed on OVA conjugated PLURONIC-stabilized nanoparticles and demonstrated that the size was maintained at  ̃25 nm. Dual labeled OVA-conjugated PLURONIC-stabilized nanoparticles were present in lymph nodes at 24 and 48 h post-injection, as demonstrated in FIG. 11. The OVA was present in the same locations as nanoparticles. These results demonstrate that functionalizing the nanoparticles with a protein antigen OVA of  ̃43 kDa MW does not significantly effect nanoparticle size. The ability to produce OVA-conjugated nanoparticles of 25 nm allows the protein antigen to be delivered via lymphatics to DCs in lymph nodes. This delivery of antigen to resident lymph node DCs offers the possibility of enhancing the subsequent adaptive immune response. OVA is presented here merely as an exemplary, model antigen. Any number of molecular antigens can be analogously utilized, including peptides, proteins, including glycopeptides, and nucleic acids that encode protein antigens.
Example 13
DC Maturation Induced by OVA Conjugated to PLURONIC-Stabilized Nanoparticles
25 nm OVA-conjugated PLURONIC-stabilized nanoparticles and OVA mixed with lipopolysaccharide (LPS) were injected intradermally into mice as described herein. Lymph nodes were then harvested and cells were isolated and stained for CD11c, CD86, CD80, and CD40 as described herein. Flow cytometry was performed to determine maturation profile of lymph node DCs.
As shown in FIG. 12, the CD86, CD80, and CD40 profiles for 25 nm OVA-conjugated PLURONIC-stabilized nanoparticles compared to positive control OVA with LPS are nearly the same. Both show high expression of these DC maturation markers. These results show that the DC maturation response in vivo is nearly the same for OVA delivered by PLURONIC-stabilized 25 nm nanoparticles and OVA co-injected with the molecular danger signal LPS. This suggests the possibility to use OVA conjugated to small nanoparticles, e.g. 20-45 nm, that are hydroxylated and complement-activating, e.g., formed by PLURONIC-stabilization of PPS nanoparticles, as both antigen delivery vehicles and maturation stimulus adjuvants.
Example 14
T Cell Proliferation
A method known as T cell adoptive transfer was used to measure T cell proliferation. OT-II Tg (Jackson Immunoresearch) mice are transgenic in that they have an upregulated level of OVA T cell receptor in CD4 T cells. Spleen and lymph nodes from OT-II Tg mice were isolated to make cell suspensions. For spleen cell suspensions, red blood cells were lysed with 1.667% NH4Cl. Cells from spleen and lymph nodes were pooled and count: total of 400×106 cell recovered
Cells were labeled with carboxyfluoroscein succinimidyl ester (CFSE) and re-suspended at 20×106/ml in RPMI w/o FBS. CFSE stock was 5 mM in DMSO. A first dilution 1/10 was made in PBS and the volume necessary was added into the cells to have a final concentration of 5 μM. CFSE was added, mixed gently and incubated with the cells at 37° C. for 10 min and left with the lid open and mixed gently about every 2 min (cells were split into 2 tubes to prevent clot formation and cell death). After incubation, RPMI with 5% FBS was added to wash the cells, washed 1× with RPMI w/o FBS and 1× with PBS. A cell count was performed after CFSE labeling for a total of 300×106 cells.
Cells were re-suspended in PBS at 50×106/ml, and 10×106 cells were injected (200 μl/mouse) in the tail vein of CD45.1 congenic recipient mice. A fraction of cells were kept to check by flow cytometry CFSE labeling and the proportion of CFSE+ T cells injected. Cells were stained with APC anti-CD4.
At day 2, 20 μl of antigen (OVA at 10 ug+5 ug LPS or 25 nm OVA-conjugated PLURONIC-stabilized nanoparticles at 10 ug) were injected into the front footpads of recipient mice. At day 5, mice were sacrificed and from each mouse, brachial and axillary lymph nodes were removed and pooled to make cell suspensions. The cells were stained for CD45.2 PE, propidium idodide (dead cells), and CD4 Allophycocyanin. Flow cytometry was performed to measure T cell proliferation.
FIG. 13 (left) shows that after an injection of PBS, all CFSE labeled OT-II T cells remained at a maximum fluorescence level. However, following injection of positive control OVA with LPS (center) and OVA conjugated to 25 nm PLURONIC-stabilized (and thus hydroxylated) PPS nanoparticles there is a significant decrease in fluorescence of CD4 T cells. This decrease in fluorescence is indicative of daughter populations of cells, which exhibit less fluorescence than parent populations. There were approximately 7 cycles of proliferation after injection of OVA with LPS or OVA conjugated to PLURONIC-stabilized PPS nanoparticles. OT-II mice are transgenic in that they possess CD4 T cells that upregulate T cell receptor for OVA. Therefore these T cells are extremely sensitive when they encounter the OVA antigen. Therefore, adoptive transfer of CFSE labeled OT-II T cells into WT mice is an excellent model for measuring T cell proliferation in vivo.
Herein it is demonstrated that lymph node DCs mature following delivery of OVA conjugated to 25 nm PLURONIC-stabilized (and thus hydroxylated) PPS nanoparticles. These results now show that following this internalization of nanoparticles, the OVA antigen is processed at least partially through the MHC-II pathway and its antigenic peptide is presented by mature DCs to CD4 T cells. The CD4 T cells in turn become activated and proliferate. Activated CD4 T cells are capable of helping adaptive immune responses (e.g., presenting antigen to B cells for the induction of antibody production). Our results demonstrate that OVA conjugated to 25 nm PLURONIC-stabilized (and thus hydroxylated and complement-activating) are capable of inducing T cell proliferation at very similar level as positive control OVA with LPS. This is significant as it suggests that further T cell mediated immune responses will be mounted.
Example 15
CD8 T Cell Memory—Measurements of Cellular Immunity
25 nm OVA-conjugated PLURONIC-stabilized nanoparticles, OVA in PBS, and OVA with LPS were injected into C57/BL6 mice as described herein. Mice were then given a booster injection at 7 d. At 21 d, mice were then sacrificed and lymph nodes were removed and cells were isolated as described herein. Cell suspensions were counted using a hemocytometer.
An ELISPOT plate for IFN-γ (eBioscience) was prepared according to manufacturer's protocol. RPMI media with 10% mouse serum was added to each well at 20 μl/well. Next 2 units of IL-2 and 0.4 μg of CD28 was added to each well. Next 20 μl/well of OVA323-339 MHC-I peptide was added at 2 mM concentration. Cells were then added to the wells at 100 μl/well. Some wells received no OVA peptide, as an unstimulated control while other wells received PMA as a positive control. The plate was kept in a 37° C. incubator for 2 days. After 2 days, ELISPOT plate was developed according to manufacturer's protocol. Images of wells were taken with Leica MZ16FA stereoscope. Spots in wells were then counted with Matlab image analysis program.
As shown in FIG. 14, following immunization of mice, an ELISPOT assay was used to determine the amount of IFN-γ producing CD8 T cells (measured by spots on the plate) after re-exposure to antigen. Mice that received injections of OVA in PBS showed very low numbers of IFN-γ T cells, while there was a significant increase in mice injected with OVA conjugated to 25 nm PLURONIC-stabilized (and thus hydroxylated and complement-activating) nanoparticles and positive control OVA with LPS. Consistent with other results presented herein, OVA conjugated to very small nanoparticles (e.g., 20-45 nm) that are complement-activating (e.g., PLURONIC-stabilized (and thus hydroxylated) PPS nanoparticles) are capable of inducing DC maturation and CD4 T cell proliferation. These results showed that OVA conjugated to PLURONIC-stabilized 25 nm nanoparticles can sufficiently produce CD8 T cell memory. CD8 T cells respond to antigen processed and presented by the MHC-I pathway. The MHC-I pathway is generally associated with antigen that is processed in the cytoplasm of DCs. This suggests that the nanoparticles can deliver antigen for both MHC-I and -II processing and presentation. CD8 T cells are also known as cytotoxic killer T cells as they directly attack pathogen and pathogen infected cells. Therefore the ability of small (e.g., 20-45 nm) complement activating (e.g., PLURONIC-stabilized) nanoparticles to produce CD8 T cell memory shows the strong potential for this to be used in vaccines.
Example 16
Antibody (Ab) Titers—Measurements of Humoral Immunity
25 nm OVA-conjugated PLURONIC-stabilized (and thus hydroxylated) nanoparticles, 100 nm OVA-conjugated PLURONIC-stabilized (and thus hydroxylated) nanoparticles, 25 nm OVA-conjugated methoxy-terminated PLURONIC-stabilized (and thus not hydroxylated) nanoparticles, OVA in PBS, and OVA with LPS were injected into C57/BL6 mice as described above. 25 nm OVA-conjugated PLURONIC-stabilized (and thus hydroxylated) nanoparticles and OVA with LPS were injected into C3−/− mice. Serum was isolated from blood taken from mice prior to injections and at 21 d post-injection and stored at −20° C. until used. There were no booster injections.
96 well plates (Becton Dickinson) were coated with OVA in PBS (2 μg/ml) at 100 μl/well. Plates were left overnight at room temperature (RT). Unbound antigen was flicked off and plate was washed 3× with 200 μl/well DI water. Plates were then blocked for 1.5 h at RT with 200 μl/well blocking buffer.
Mouse serum samples were diluted serially from 1:103 until 1:108 in blocking buffer. When plates were finished blocking, they were washed 3× with 150 μl/well wash buffer. Serum samples were then added to wells at 50 μl/well for 2 h at RT. Pre-injected serum samples were added in triplicated. Samples were then flicked out and plate was then washed with wash buffer 5× at 150 μl/well. Mouse anti-IgG-HRP was diluted in blocking buffer 1:3000 and added at 50 μl/well for 2 h at RT. HRP antibody was then flicked out and plate was washed with wash buffer 5× at 150 μl/well. Next, HRP substrate reagent (R&D systems) was added at 100 μl/well in the dark for 45 min at RT. The reaction was stopped by adding 50 μl/well of 2N H2SO4. Plate absorbance was then measured on Tecan plate reader at 450 nm and 540 nm wavelength. The 540 nm background values were subtracted from 450 nm to obtain final values. A positive sample was determined if the post-injected serum value was greater than a cutoff value. The cutoff value was calculated from the pre-injected triplicate average plus standard deviation multiplied by 3. The highest dilution with a positive value is considered the antibody (Ab) titer value.
The log10 IgG OVA Ab titers were determined in mice injected with various treatments, as shown in FIG. 15. Mice injected with the negative control of OVA with PBS showed no positive titers. Mice injected with the positive control of OVA with LPS showed positive titers between 3-6 in both wild-type and C3−/− mice. Injections of OVA conjugated to 25 nm PLURONIC-stabilized PPS nanoparticles produced titers of 4, while injections of OVA conjugated to 100 nm PLURONIC-stabilized PPS nanoparticles and OVA conjugated to 25 nm methoxy-terminated PLURONIC-stabilized PPS nanoparticles produced lower titer values. Finally Ab titers of the animals treated with OVA conjugated to 25 nm PLURONIC-stabilized PPS nanoparticles was significantly lower in C3−/− mice.
The presence of OVA IgG Ab titers is evidence of humoral immunity. One route for this process to occur is when antigen is processed by DCs and presented to CD4 T cells, which then stimulate B cells to produce Abs against the antigen. Delivery of free protein antigen in the absence of danger signals is known to not be capable of significantly inducing humoral immunity, and our results show this as OVA in PBS does not produce positive titers. A positive control of OVA with LPS however shows significant level of titers in both wild-type and C3−/− mice. We have shown herein that OVA conjugated to 25 nm PLURONIC-stabilized (and thus hydroxylated and complement-activating) PPS nanoparticles can induce DC maturation, T cell proliferation, and CD8 T cell memory. These results show that OVA conjugated to 25 nm PLURONIC-stabilized (and thus hydroxylated and complement-activating) nanoparticles can also induce humoral immunity by production of anti-OVA IgG titers. OVA conjugated to 100 nm PLURONIC-stabilized nanoparticles do not produce positive titers, demonstrating that lymph node targeting is crucial for nanoparticle induced humoral immunity. Also OVA conjugated to 25 nm methoxy-terminated PLURONIC-stabilized nanoparticles, which activate complement to a much lower extent, show reduced Ab titer values, proving that control of the surface chemistry is also necessary to produce such a strong immune response, mediated through complement activation. Finally it is shown that complement activation is playing a significant role in the induction of humoral immunity as OVA conjugated to 25 nm PLURONIC-stabilized PPS nanoparticles produce much lower titer values in C3−/− mice than in wild-type mice.
These results prove that nanoparticles of the various embodiments that are made as described herein that have both special size for working through lymph node targeting, and special surface chemistry, i.e., capable of complement activation (e.g., hydroxylated, obtainable by PLURONIC stabilization and other means), can be used to produce strong T cell-dependant humoral immunity with a coinjected antigen. What is demonstrated here is the case when the antigen is conjugated to the nanoparticle surface; adsorption methods for binding the antigen to the nanoparticle surface will also be effective. Coinjection of antigen with such nanoparticles should also be effective, although perhaps to a lesser extent.
Example 17
Hydrophobic Drug Loading
Loading of hydrophobic drugs, for example dexamethasone was achieved, by an adapted solvent evaporation method [132,133]. Briefly, and by way of example, the drug was added to the solvent dichloromethane (1 mg/ml). 1 ml of drug-solvent suspension was then added to 1 ml of PPS nanoparticle solution at 20 mg/ml. The emulsions were continuously stirred in the dark at room temperature in order to evaporate the solvent. Drug loading efficiency was measured by GPC.
Example 18
Complement-Activating Nanoparticles can be Made that Induce a Th1 Biased Response Indicating Activation of Cellular Immunity
Cellular immunity, characterized by a Th1 biased response, is needed for vaccines for a number of applications. An in vitro model of activation of bone marrow-derived DCs (BMDCs) was used to gather evidence of a Th1 response by determining molecular markers of DC activation. The patterns of cytokine expression pointed to the mechanisms of action of the nanoparticle formulation. Specifically, BMDCs were exposed to nanoparticle and control formulations in vitro, and IL-12p40, a key regulator in the DC's ability to activate T cells, was measured. Robust IL-12p40 secretion was observed, comparable to that induced by exposure to LPS (FIG. 16 panel a). Consistent with the apparent mechanism of action of the hydroxyl-bearing nanoparticles, this effect was only observed after exposure of the nanoparticles to serum (as would happen after injection), and it was dependent upon the presence of hydroxyl groups on the nanoparticle surface, to activate complement. Such a strong IL-12p40 response shows a full response of the adaptive immune response including a Th1 response. FIG. 16 panel b shows that MHCI increased four-fold in the presence of serum for nanoparticles displaying hydroxyl groups compared to serum alone. Further, FIG. 16 panel c shows that 25 nm, PPS-core, complement-activating nanoparticles with hydroxyls induced CD8 T cell memory in mice, as evidenced by interferon-gamma production caused by the nanoparticles but not controls.
Example 19
Complement-Activating Nanoparticles Display an Advantageous Safety Profile
These results surprisingly demonstrate that the route of complement activation for provision of an activation signal for DCs has particular toxicological advantages: BMDCs stimulated in vitro with 25 nm, PPS-core, complement-activating nanoparticles after serum exposure were strongly activated (as measured by expression of CD86, CD80 and CD40), but did not express TNF-a or IL-6, cytokines that can lead to toxic effects from danger signal exposure. As adjuvants for comparisons, LPS was selected (too toxic for clinical use) and CpG DNA was selected (in active development for clinical use). This data with BMDCs demonstrates no measurable production of either TNF-a or IL-6, in contrast to that observed after exposure to CpG DNA and LPS (FIG. 17).
Example 20
Complement-Activating Nanoparticles that Function through TLR-4 Activation
Toll-like receptor TLR4 is believed to mediate the potent response of nanoparticle-bound complement. When IL-12p40 secretion from BMDCs isolated from TLR4−/− mice (mice that do not express TLR4) was measured for 25 nm, PPS-core, complement-activating hydroxylated nanoparticles exposed to serum, the IL-12p40 levels are substantially depressed when compared to wild-type mice (which express TLR4); as control, BMDCs stimulated with CpG DNA (a ligand for a different TLR) demonstrated no difference (FIG. 18 panel a). Moreover, when anti-ovalbumin (OVA) titers were measured for hydroxylated nanoparticles, the OVA-bound nanoparticles failed to induce a response in TLR4−/− animals, whereas the control adjuvant CpG DNA did induce a normal response (FIG. 18, panel b). Mice without TLR4 did not create antibodies against the OVA-bound nanoparticles but mice with TLR4 receptors did generate anti-OVA antibodies in response to OVA-bound nanoparticles. Control biological and chemical measurements have decisively ruled out the possibility that the nanoparticles are contaminated with a TLR-activator.
Example 21
Nanoparticle-Bound Thiol Moieties Trigger an Adaptive Immune Response
Nanoparticles with surface thiols have unexpected and surprising effects on activation of a complement-dependent adaptive immune response. These results point to new nanoparticle materials formulations and uses. For example, 25 nm, PPS-core nanoparticles bearing surface thiols, either with hydroxyls or methoxyls also present on the surface, potently activated complement as measured by C3a production, whereas the absence of thiols significantly reduced the level of complement activation (FIG. 19). This phenomenon was most potent with nanoparticle surfaces having both hydroxyls and thiols.
Additionally, in vitro BMDCs were activated strongly (measured by CD80 expression and IL-12p40 production) with 25 nm, PPS-core nanoparticles bearing a hydroxy and thiol surface. However this effect was not observed with a nanoparticle hydroxy surface absent of thiols (FIG. 20).
Example 22
Molecular Danger Signals can be Incorporated in Nanoparticle Vaccine Compositions
Various molecular danger signals can be incorporated into or upon the nanoparticle surfaces. While it is possible to attach hydrophilic danger signals on the surface of the nanoparticles, it is also possible to incorporate hydrophobic danger signals within the hydrophobic core of the nanoparticles. An example of a hydrophobic danger signal is the drug imiquimod, and members of its family, which activate TLR-7. Imiquimod was loaded within the hydrophobic cores of 25 nm PPS-core nanoparticles as already described, and these were exposed to BMDCs in vitro. Characterization of DC activation by flow cytometry provided strong evidence of DC activation by the imiquimod incorporated in the hydrophobic core of nanoparticles (FIG. 21).
Example 23
Self-Assembled Nanoparticles as a Technology Platform
Micellar Formation
Nanoparticles, including polymer micelles, are available to implement a variety of embodiments for complement activation. FIG. 22 shows an example of a route to synthesis of OH-PEG-PPS with or without blockade of a terminal —SH group. FIG. 23 shows an example of a route to synthesis of CH3-PEG-PPS. These molecules may be used to form micelles. Methods of micelle formation include dispersion in aqueous media from organic solvents, suspension in aqueous media from thin films, or direct hydration of polymer.
An example of methods of micelle formation may be found in Velluto et al., Molecular Pharmaceutics 2008, “PEG-b-PPS Diblock Copolymer Aggregates for Hydrophobic Drug Solubilization and Release: Cyclosporin A as an Example”, which is hereby incorporated by reference. Certain embodiments include micelles loaded with an agent at an efficiency of more than about 30%; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., more than about 50%, or about 66%. Loading methods include, for example, a cosolvent evaporation method or suspension in hot water. Agents, e.g., antigens, drugs, or nucleic acids, may be encapsulated in the micelles. Encapsulation methods include those with efficiencies of more than about 30%; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., more than about 50%, or about 70%.
Micellar Formation with Antigen Attachment
Antigen can also be readily attached to PEG-PPS block copolymer micelle surfaces. One route by which to do this is to provide some chain termini with an amine functionality, by which to then couple a peptide or protein antigen. One route to such a polymer is shown in FIG. 24.
Other routes for coupling antigen have also been carried out. One scheme that has been used is to synthesize the peptide or protein antigen to bear an unpaired cysteine residue, which can be attached to a pyridyl disulfide group on the nanoparticle surface, as shown in FIG. 25. Another approach has been used is to synthesize the peptide or protein antigen to comprise a protease substrate that is involved in antigen processing, such as a cathepsin S substrate; cathepsin S is involved in both MHC I and MHC II antigen processing. The sequence KQKLR (SEQ ID NO:3) is described as an example. In this way, the actual antigenic peptide is generated in situ.
Schemes as shown in FIG. 25A, and also in an alterative arrangement in FIGS. 25B and 25C, have been used to conjugate peptide antigens to nanoparticle surfaces and have been demonstrated to release their peptide payload intracellularly after cellular uptake and processing. For example, FIG. 26 shows results wherein the SIINFEKL (SEQ ID NO: 2) MHC I peptide of ovalbumin was been conjugated to 25 nm, PPS-core, complement-activating hydroxylated nanoparticles, and after injection and harvesting of lymph node-resident DCs was demonstrated by flow cytometry to display the peptide correctly within the MHC I complex, using an antibody specific to the SIINFEKL-MHC I complex. This demonstrates both that the conjugation scheme is effective and also that the designed release mechanism, namely reduction of the disulfide, functions well.
Schemes have been developed by which to add thiol functionality to antigens that may not have such a functionality, for example protein antigens when one does not wish to express a variant antigen bearing an unpaired cysteine. The terminal alpha-amine may be modified in many cases to a terminal thiol using the scheme presented in FIG. 27, for example. Experimentation with DC activation has demonstrated that the same effects that have been observed with crosslinked-core PPS nanoparticles are also seen in PEG-PPS micelles. Extraction, and cell culture of bone marrow-derived dendritic cells (BMDCs,) from 6-8 week old C57BL6/J healthy mice was accomplished, using recombinant GM-CSF to induce differentiation into DCs. Micelles of PEG-PPS were prepared as follows. To form the non-thiol micelles, micelles were formed from THF using H3CO-EG46-PS17-phth, HO-EG68-PS20-phth, H3CO-EG46-PS11—SH, and HO-EG68-PS25—SH in 10 mmol PBS pH 7.4 (phth indicating phthalimidyl). The samples were standardized to 8.2 mg/mL for H3CO-EG46-PS17-phth and H3CO-EG46-PS11—SH, and 16.6 mg/mL for HO-EG68-PS25—SH and HO-EG68-PS20-phth. These were then added to BMDCs (100,000 cells per well,) at 122 μL for the H3CO polymers or 60.3 μL for the HO polymers to add 1 mg of polymer per well. The cells and micelles were then incubated either with or without mouse serum for 45 min, washed, and then incubated in media for 24 h. The cells were labeled with anti-CD11c-allophycocyanin and anti-CD86-PE (Pharmigen) antibodies and results were quantified as above using FACS. Pluronic F-127 PPS core nanoparticles ( ̃1 mmol SH,) and unmethylated CpG controls were used for comparison.
The micelles used in this example were 18 nm average diameter and can be easily made within the range of 10 nm-50 nm by adjusting the PEG and PPS molecular weights. The artisan will recognize that the shape of the micelle or self-assembled structure will depend on the ratio of the hydrophilic block molecular weight (here the PEG molecular weight) to the hydrophobic block molecular weight (there the PPS molecular weight), whereas the size of the micelle or self-assembled structure will depend on the total molecular weight. Thus, larger spherical micelles than those examined in this example can be derived from higher molecular weight block copolymers at similar block molecular weight ratio and smaller spherical micelles can be derived from lower molecular weight block copolymers at similar block molecular weight ratios. It is noted that with the PEG-PPS micelles shown in this example, the same importance of HO-groups as well as a role for HS— groups via complement activation was observed, as shown in FIG. 28. As such, the micellar implementation of antigen-bearing, complement activating, lymph-node targeting micelles is very useful.
In addition to antigen molecules, danger signal molecules can also be chemically conjugated to the nanoparticle surface using the chemical schemes described above. For example, as shown in FIG. 29, CpG DNA can be coupled to the nanoparticle surfaces.
Example 24
Branched or Multi-Functional Soluble Polymers are a Useful Platform
These data show that it is possible to form very small nanostructures capable of activating complement and displaying an antigen from soluble multifunctional polymers, such as branched polymers, for instance, branched PEGs. The ability of branched PEGs, which are hydroxylated and capable of complement activation, to activate DCs in the lymph nodes in vivo was established by injecting 8-arm PEG of 40 kDa molecular weight. Cells were isolated from the lymph nodes of mice, and DCs were analyzed for activation by flow cytometry. As shown in FIG. 30, HO-terminated branched PEG was able to potently activate DCs in vivo. Schemes are available to generate branched PEGs that are also activated for antigen coupling, such as that shown in FIG. 31.
Example 25
Nanoparticle Vectors for DNA Vaccines and Other Applications
It has been demonstrated that the ultrasmall DNA complexes formed from PEG-PPS blended with PEG-PPS-PEI (see WO2007/008300 or US2005000686188, which are hereby incorporated by reference herein), which are very small in dimension (as shown there, 30 nm diameter), can enter the lymphatics after interstitial injection and can effectively transfect cells in the lymph node. A scheme for synthesis of PEG-PPS-PEI, which also referred to as PEG-PPS-ss-PEI to indicate the presence of an disulfide link between the PPS domain and the PEI domain (this disulfide link is optional) is shown in FIG. 32A. This Figure illustrates synthesis with a PEG of degree of polymerization of 44, but other degrees of polymerization can be handled in the same manner. Micelle size can be powerfully controlled by forming mixed micelles from a blend of PEG-PPS-PEI and PEG-PPS, as illustrated in this example. When mixed micelles were formed from PEG44-PPS40-PEI120 (1 mg) and PEG44-PPS20 (10 mg), complexes with DNA were only 30 nm in average diameter, which the artisan will recognize as being a very small DNA complex. Thus, although rather large micelles were formed from PEG-PPS-PEI of the molecular weight shown, blending with PEG-PPS could drive micelle size to much smaller dimensions, even after complexation with DNA. As shown in FIG. 32B, in two separate experiments, greater then 5% of the cells resident in the lymph node were transfected with green fluorescent protein (GFP), a remarkably high number. Of these, flow cytometric analysis showed T cells and B cells to be very effectively transfected, and DCs were also effectively transfected. The concept of DNA vaccines, i.e. providing the antigen encoded as a genetic sequence, has of course been demonstrated by others, however the artisan will recognize that transfecting cells in the lymph node as described herein, rather than cells in the periphery, is a very different concept that leads to new therapies, new therapeutic approaches, and requires distinct devices. Thus, the ultrasmall nanoparticulate forms described herein that have a structure capable of targeting the lymph nodes by interstitial flow and transfecting immune cells in the lymph node, such as B cells, DCs and macrophages, as well as T cells, presents a powerful new approach to DNA vaccines. This is enabled by provision of DNA as very small complexes, less than about 50 nm, and more preferably about 30 nm.
Example 26
Nanoparticle Vectors for Antigen-Specific Tolerogenic Vaccines
Nanoparticles and polymer micelles as described herein can be used to deliver antigens to dendritic cells and also deliver immunosuppressants to the cells. FIG. 33 shows that bone marrow-derived DCs can be exposed to dexamethasone-loaded 25 nm PPS-core nanoparticles, and then can be exposed to very strong DC activation signals such as LPS; the nanoparticle-incorporated immunosuppressant prevents DC activation even to such a strong signal.
Example 27
Physicochemical Mechanisms of Antigen Incorporation
In addition to covalent grafting of antigen on the nanoparticle, physicochemical means are also contemplated. One powerful method is synthesis or expression of a peptide or protein antigen to incorporate a strongly anionic stretch of amino acids, such as an oligoE or oligoD region, to enable formation of an electrostatic complex, as for siRNA or aptamer complexes. Just as complexes of polyanionic DNA with PEG-PPS-PEI-containing micelles can be formed, it is possible to incorporate antigens comprising engineered anionic regions into these polycation-containing polymer micelles. Another physicochemical method is to incorporate the antigen peptide or protein within watery-core vesicles, for example formed with PEG-ss-PPS. The formation of such vesicles may be accomplished, see Cerritelli et al., Biomacromolecules 2007:1966-1972. When cytoplasmic release of antigen is desired, as in the case of MHC I presentation, this method of antigen delivery is particularly useful. The size of such vesicles can be controlled by a number of means, for example by perfusion through membrane filters producing vesicles of similar size as the pores of the membrane through which they are perfused, see Napoli et al., Nature Materials, 2004, 3:183-189. Release after cellular uptake can very effectively deliver a water-soluble payload to the cytoplasm of DCs, as demonstrated with bone marrow-derived DCs in FIG. 34. Accordingly, embodiments of the invention include nanoparticles that are watery-core vesicles or vesicles, e.g., nanoparticles in a size range of about 10 nm to about 50 nm or 70 nm; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., less than about 50 nm or 18 nm.
Example 28
Delivery of Nanoparticles to Mucosal Surfaces
In the examples above, the main route of administration explored was injection in tissues, such as intradermal or subcutaneous injection, although intramuscular injection would be expected to be similar. The ultrasmall size of the nanoparticles was key to entry of the nanoparticles in to the draining lymphatics. The ultrasmall nature of nanoparticles can also be used to gain entry into other tissues. In vaccination, the mucosa is interesting, in that it is desirable to generate a mucosal immunity, and also in that needle-free administration can be obtained. The mucosa can be accessed within the mouth, after injection, in the nasal cavity, in the vaginal cavity, and in the rectal cavity. PPS-core nanoparticles, 30 nm in diameter, were administered to the sublingual mucosa in the rodent, and penetration into the mucosal tissue was determined by fluorescence microscopy. Furthermore, the nanoparticle formulations were administered to the sublingual mucosa under mild pressure with the aid of a blunt-ended cannula. FIG. 35 demonstrates that 30 nm PPS-core nanoparticles penetrated the sublingual mucosa extremely effectively under mild pressure, whereas 100 nm PPS-core nanoparticles did not penetrate nearly as effectively. Penetration of 100 nm PPS-core nanoparticles was much less than that obtained with 30 nm PPS-core nanoparticles. These results demonstrate that ultrasmall nanoparticles can be useful in penetrating across tissue barriers, such as the mucosa, in addition to permeation within tissue barriers, such as the interstitium. In the case of penetration of the mucosa, including the oral mucosa, both penetration into the mucosal layer, for collection by antigen presenting cells here, and penetration across the mucosal layer for eventual collection in the draining lymph nodes are interesting. FIG. 35 demonstrates penetration into the depth of the mucosa when very small, 30 nm nanoparticles were administered along with a mild pressure. Although a blunt-tipped cannula was used in this example in the rodents, in man very simple devices, such as elastic or otherwise deformable cups pressed with their open side upon the sublingual tissue with the aid of the finger can be used to apply the mild, penetration-enhancing pressure.
Statistics
Statistical significance in the Examples was determined by performing a two-tailed Student's t test unless otherwise indicated. Results indicate mean±SD and 3-8 experiments were performed for each condition unless otherwise indicated.
Carriers, Diluents, Kits
Pharmaceutically acceptable carriers or excipients may be used to deliver embodiments as described herein. Excipient refers to an inert substance used as a diluent or vehicle for a therapeutic agent. Pharmaceutically acceptable carriers are used, in general, with a compound so as to make the compound useful for a therapy or as a product. In general, for any substance, a pharmaceutically acceptable carrier is a material that is combined with the substance for delivery to an animal. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. In some cases the carrier is essential for delivery, e.g., to solubilize an insoluble compound for liquid delivery; a buffer for control of the pH of the substance to preserve its activity; or a diluent to prevent loss of the substance in the storage vessel. In other cases, however, the carrier is for convenience, e.g., a liquid for more convenient administration. Pharmaceutically acceptable salts of the compounds described herein may be synthesized according to methods known to those skilled in this arts. Residual amines may be complexed with sodium, potassium, or a other suitable counter-ions. Thus a pharmaceutically acceptable composition has a carrier, salt, or excipient suited to administration to a patient. Moreover, inert components of such compositions are biocompatible and not toxic.
The nanoparticles described herein may be administered in admixture with suitable pharmaceutical diluents, solutions, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. Thus the deliverable compound may be made in a form suitable for oral, rectal, topical, intravenous injection, intra-articular injection, or parenteral administration. Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used. Suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents may be included as carriers, e.g., for pills. For instance, an active component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. The compounds can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The active compounds can also be administered parentally, in sterile liquid dosage forms. Buffers for achieving a physiological pH or osmolarity may also be used. In use, nanoparticles may be provided to a patient by a method comprising introducing to the patient, e.g., by injection, orally, buccaly, as a suppository, transdermally, transdermal patch, or topically, e.g., by ointment or salve.
Embodiments may be prepared as part of a kit with components for preparing and/or delivering the nanoparticles. A kit refers to a collection of materials needed by a user to accomplish the intended task; the kit may be provided, for instance, in a unitary housing such as a box, envelope, or package. Instructions to prepare and/or use the nanoparticles may include steps as needed to guide the user, e.g., as described elsewhere herein. An applicator may be provided in the kit or separately. Examples of applicators are syringes, swabs, wipes, or devices to create mild pressure.
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## Claims

1. A medical composition for introduction into a mammal comprising:
a collection of synthetic nanoparticles that have a mean particle diameter of less than about 70 nm, wherein the nanoparticles comprise an antigen and a synthetic polymer with functional groups disposed on an exterior of the nanoparticle that activate complement in a mammal wherein the antigen is bound to the synthetic polymer with a disulfide bond, and the synthetic polymer comprises a polymer selected from the group consisting of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol), poly(propylene sulfide), and an amphiphillic poly(propylene sulfide) block copolymer.

2. The composition of claim 1, wherein the functional groups comprise nucleophilic groups.

3. The composition of claim 1, wherein the functional groups comprise hydroxyls and/or thiols.

4. The composition of claim 1, wherein the nanoparticles are micelles.

5. The composition of claim 4, further comprising a therapeutic agent encapsulated with the micelle.

6. The composition of claim 1, wherein the composition is free of an adjuvant other than the synthetic polymer.

7. The composition of claim 1, wherein the functional groups induce a Th-1 response in a mammal.

8. The composition of claim 1, wherein antigen is a peptide conjugated to the nanoparticles by the disulfide bond.

9. The composition of claim 1, wherein the collection further comprises a biomolecule danger signal conjugated to the nanoparticles by a further disulfide bond.

10. The composition of claim 1, further comprising a hydrophobic danger signal.

11. The composition of claim 1, wherein the antigen is a tumor antigen or an antigen of an infectious disease.

12. The composition of claim 1, wherein the nanoparticles further comprise a danger signal selected from the group consisting of inflammatory cytokines and ligands for Toll-like receptors.

13. The composition of claim 1, wherein the nanoparticles are free of targeting ligands that specifically bind to a cell, the antigen is an antigen for tumor immunotherapy or infectious disease, and further comprising a danger signal chosen from the group consisting of inflammatory cytokines and ligands for Toll-like receptors.

14. The composition of claim 1, wherein the synthetic polymer comprises a hydrophobic core and a hydrophilic corona, wherein the core is comprised of poly(propylene sulfide) and the hydrophilic corona is comprised of polyethylene glycol, and wherein the functional groups comprise nucleophiles.

15. The composition of claim 14, wherein the nucleophiles comprise hydroxyls and/or thiols and the antigen comprises Caspase-8, MAGE-1, Tyrosinase, HER-2/neu, MUC-1 or survivin.

16. The composition of claim 1, wherein the nanoparticles further comprise an immunosuppressant.

17. An immunostimulatory composition for introduction into a patient that comprises a collection of synthetic nanoparticles that have a mean particle diameter of less than about 70 nm, wherein the nanoparticles comprise an antigen and a synthetic polymer, wherein the antigen is bound to the synthetic polymer with a disulfide bond, and the synthetic polymer comprises a polymer selected from the group consisting of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol), poly(propylene sulfide), and an amphiphillic poly(propylene sulfide) block copolymer, with the composition inducing an immune response against the antigen in the patent without inducing a TNF-alpha response and without inducing an IL-6 response.

18. The composition of claim 17, wherein the nanoparticles are free of targeting ligands that specifically bind to a cell, and the antigen is an antigen of a tumor or an infectious disease.

19. The composition of claim 17, wherein the nanoparticles further comprise an immunosuppressant.

20. The composition of claim 17, wherein the synthetic polymer comprises a core and a hydrophilic corona, wherein the core is comprised of poly(propylene sulfide) and the hydrophilic corona is comprised of polyethylene glycol.

21. A method of transfecting a cell in a lymph node of a patient comprising:
administering to a mucosal surface of a patient, under mild pressure, a collection of synthetic nanoparticles that have a mean particle diameter of less than about 70 nm, wherein the nanoparticles comprise an antigen and a synthetic polymer, wherein the antigen is bound to the synthetic polymer with a disulfide bond, and the synthetic polymer comprises a polymer selected from the group consisting of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol), poly(propylene sulfide), and an amphiphillic poly(propylene sulfide) block copolymer.

22. A method of inducing a Th1-mediated immunological response to an antigen that involves toll-like receptor TLR4 in a patient without inducing a TNF-alpha response and without inducing an IL-6 response comprising
introducing into the patient a pharmaceutically acceptable composition comprising a collection of synthetic nanoparticles that
have a mean particle diameter of less than about 70 nm and
comprise the antigen and a synthetic polymer, wherein the antigen is bound to the synthetic polymer with a disulfide bond, and the synthetic polymer comprises a polymer selected from the group consisting of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol), poly(propylene sulfide), and an amphiphillic poly(propylene sulfide) block copolymer,
with the composition inducing an immune response against the antigen in the patent without inducing a TNF-alpha response and without inducing an IL-6 response.

23. The method of claim 22, wherein the nanoparticles are free of targeting ligands that specifically bind to a target cell, and the antigen is an antigen of a tumor or an infectious disease.

24. The method of claim 22, wherein the nanoparticles comprise a core and a hydrophilic corona, wherein the core is comprised of poly(propylene sulfide) and the hydrophilic corona is comprised of a synthetic polymer that comprises polyethylene glycol and nucleophilic functional groups.

25. The method of claim 22, wherein the nanoparticles further comprise an immunosuppressant.

26. The composition of claim 1, wherein the synthetic polymer is an amphiphillic poly(propylene sulfide) block copolymer.