Patent Description:
Ultraviolet (UV) radiation from sunlight can lead to multiple adverse effects including cutaneous phototoxicity (sunburn), photoaging, and carcinogenesis (<NPL>); <NPL>)). UVB directly induces cyclopyrimidine dimers (CPDs) within the genomic DNA (gDNA) of keratinocytes, and both UVA and UVB exposure markedly enhance production of reactive oxidation species (ROS) that damage a variety of cellular components, including gDNA (<NPL>)), and induce immunosuppressive cytokines (<NPL>)). UV-exposure is clearly linked to both melanoma and non-melanoma skin cancer development (<NPL>)). Over the past few decades, commercially available UV-protective sunblocks have largely incorporated organic UV filters [e.g. avobenzone, octinoxate, octocrylene, oxybenzone and padimate O (PO) (<NPL>)] as formulations based on oil/water emulsions (<NPL>)). There are substantial concerns, however, that these aromatic organic compounds can penetrate through the stratum corneum, or via follicles, into epidermal cells, keratinocytes and Langerhans cells (<NPL>)). The potential for systemic absorption of such organic compounds, and their depot in adipose tissue, has also been a concern (<NPL>); <NPL>); <NPL>); <NPL>.

Alternatively, UV-blocking inorganic materials such as micronized zinc oxide (ZnO) and titanium dioxide (TiO<NUM>) particles (<NPL>)) have been utilized. While transdermal penetration of the inorganic particles appears to be less of a concern than for the organic agents, both types of sunblock agents have shown the capacity to enhance ROS generation after UV exposure, suggesting even small quantities may contribute to cellular damage and ultimately carcinogenesis (<NPL>); <NPL>); <NPL>); <NPL>)). Thus, while the application of such products protects against sunburn, e.g. raises the skin's minimal erythema dose (MED), there continues to be controversy regarding their overall effectiveness in preventing skin cancer (<NPL>); <NPL>); <NPL>); <NPL>)). Moreover, several UV filters have been detected in human urine and breast milk samples after tropical treatment, and may mediate systemic effects including endocrine disruption (<NPL>); <NPL>); <NPL>)). Therefore, preventing direct skin contact and subsequent epidermal penetration may be essential to eliminating the potential adverse effects of sunscreens.

Some commercially available sunscreens are opaque, due to their use of large particles (<NPL>)). The smaller, non-adhesive nanoparticles used in other commercially available sunscreens accumulate in hair follicles or penetrate deep into dermis, causing a variety of adverse effects (<NPL>); <NPL>)). Numerical simulations of nanoparticle properties suggest that unless small nanoparticles can be clearly demonstrated as safe, it is increasingly difficult to solve this paradox (<NPL>)). (<NPL>); <NPL>); <NPL>); <NPL>)).

Commercial sunscreens polymerize monomers with an initiator in order to stabilize the UV filters into a film that coats the skin. The chemicals involved include a variety of acrylate derivatives and multiple initiators (<NPL>), which have been implicated in irritant and allergic contact dermatitis (<NPL>); <NPL>)).

It is therefore an object of the present invention to provide improved sunblock particles for use in sunscreens.

<CIT> discloses nanoparticles comprising a core, which consists of an inorganic material, and a shell, which consists of a linker and dendritic polyglycerol sulfate.

<NPL>) discloses nanoparticles comprising poly(lactic acid-co-glycolic acid) conjugated with hyperbranched polyglycerol and surface-modified with a transferrin antibody.

<NPL>) discloses nanoparticles comprising polylactic acid grafted onto hyperbranched polyglycerol and loaded with BSA protein.

Core-shell particles, such as microparticles and nanoparticles, and methods of making and using thereof are described herein. The core contains a hydrophobic polymer. The shell contains hyperbranched polyglycerol (HPG). The HPG is covalently bound to the hydrophobic polymer such that the hydrophilic HPG is oriented towards the outside of the particles and the hydrophobic polymer oriented to form the core.

The HPG coating is modified to adjust the properties of the particles. Unmodified HPG coatings impart stealth properties to the particles which resist non-specific protein absorption. Hydroxyl groups on the HPG coating are chemically modified to form aldehyde functional groups that react with functional groups on tissue or otherwise interact with tissue to adhere the particles to the tissue, cells, or extracellular materials, such as proteins. Polyethylene glycol is covalently attached the surface of the particles by reaction of the aldehyde groups.

Topical formulations for application to the skin can contain these HPG coated nanoparticles. Nanoparticles with unusually strong bioadhesive properties do not diffuse into hair follicles and are useful as sunscreens or delivery of therapeutic, prophylactic, diagnostic or nutraceutical agents. In some embodiments, the adhesive particles include prophylactic agents, such as ultraviolet (UV) light filters. The particles are applied topically. They can be used as or formulated in a sunscreen. The nanoparticles are useful in other topical applications, such as transdermal delivery of therapeutic, diagnostic, nutraceutical and/or prophylactic agents.

"Effective amount" or "therapeutically effective amount", as used herein, refers to an amount of drug effective to alleviate, delay onset of, or prevent one or more symptoms of a disease or disorder.

The terms "treating" or "preventing", as used herein, can include preventing a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

"Topical administration", as used herein, means the non-invasive administration to the skin, orifices, or mucosa. Topical administrations can be administered locally, i.e. they are capable of providing a local effect in the region of application without systemic exposure. Topical formulations can provide systemic effect via absorption into the blood stream of the individual. Topical administration can include, but is not limited to, cutaneous and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ophthalmic administration, and rectal administration.

The terms "bioactive agent" and "active agent", as used interchangeably herein, include, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body. A bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

"Biocompatible" and "biologically compatible", as used herein, generally refer to materials that are, along with any metabolites or degradation products thereof, generally non-toxic to the recipient, and do not cause any significant adverse effects to the recipient. Generally speaking, biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient.

The term "biodegradable" as used herein, generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject. The degradation time is a function of composition and morphology. Degradation times can be from hours to weeks.

The term "pharmaceutically acceptable", as used herein, refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration. A "pharmaceutically acceptable carrier", as used herein, refers to all components of a pharmaceutical formulation which facilitate the delivery of the composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

The term "molecular weight", as used herein, generally refers to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (Mw) as opposed to the number-average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

The term "small molecule", as used herein, generally refers to an organic molecule that is less than about <NUM>/mol in molecular weight, less than about <NUM>/mol, less than about <NUM>/mol, less than about <NUM>/mol, or less than about <NUM>/mol. Small molecules are non-polymeric and/or non-oligomeric.

The term "copolymer" as used herein, generally refers to a single polymeric material that is comprised of two or more different monomers. The copolymer can be of any form, such as random, block, graft, etc. The copolymers can have any endgroup, including capped or acid end groups.

"Hydrophilic," as used herein, refers to the property of having affinity for water. For example, hydrophilic polymers (or hydrophilic polymer segments) are polymers (or polymer segments) which are primarily soluble in aqueous solutions and/or have a tendency to absorb water. In general, the more hydrophilic a polymer is, the more that polymer tends to dissolve in, mix with, or be wetted by water.

"Hydrophobic," as used herein, refers to the property of lacking affinity for, or even repelling water. For example, the more hydrophobic a polymer (or polymer segment), the more that polymer (or polymer segment) tends to not dissolve in, not mix with, or not be wetted by water.

Hydrophilicity and hydrophobicity can be spoken of in relative terms, such as, but not limited to, a spectrum of hydrophilicity/hydrophobicity within a group of polymers or polymer segments. In some embodiments wherein two or more polymers are being discussed, the term "hydrophobic polymer" can be defined based on the polymer's relative hydrophobicity when compared to another, more hydrophilic polymer.

The term "lipophilic", as used herein, refers to compounds having an affinity for lipids.

The term "amphiphilic", as used herein, refers to a molecule combining hydrophilic and lipophilic (hydrophobic) properties.

"Nanoparticle", as used herein, generally refers to a particle having a diameter, such as an average diameter, from about <NUM> up to but not including about <NUM> micron, preferably from <NUM> to about <NUM> micron. The particles can have any shape. Nanoparticles having a spherical shape are generally referred to as "nanospheres".

"Microparticle", as used herein, generally refers to a particle having a diameter, such as an average diameter, from about <NUM> micron to about <NUM> microns, preferably from about <NUM> to about <NUM> microns, more preferably from about <NUM> to about <NUM> microns, most preferably from about <NUM> micron to about <NUM> microns. The microparticles can have any shape. Microparticles having a spherical shape are generally referred to as "microspheres".

"Mean particle size" as used herein, generally refers to the statistical mean particle size (diameter) of the particles in a population of particles. The diameter of an essentially spherical particle may refer to the physical or hydrodynamic diameter. The diameter of a non-spherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering.

"Monodisperse" and "homogeneous size distribution", are used interchangeably herein and describe a population of nanoparticles or microparticles where all of the particles are the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which <NUM>% or more of the distribution lies within <NUM>% of the median particle size, more preferably within <NUM>% of the median particle size, most preferably within <NUM>% of the median particle size.

"Branch point", as used herein, refers to a portion of a polymer-drug conjugate that serves to connect one or more hydrophilic polymer segments to one or more hydrophobic polymer segments.

The term "targeting moiety", as used herein, refers to a moiety that binds to or localizes to a specific locale. The moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. The locale may be a tissue, a particular cell type, or a subcellular compartment. The targeting moiety or a sufficient plurality of targeting moieties may be used to direct the localization of a particle or an active entity. The active entity may be useful for therapeutic, prophylactic, or diagnostic purposes.

The term "reactive coupling group", as used herein, refers to any chemical functional group capable of reacting with a second functional group to form a covalent bond. The selection of reactive coupling groups is within the ability of the skilled artisan. Examples of reactive coupling groups can include primary amines (-NH<NUM>) and amine-reactive linking groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these conjugate to amines by either acylation or alkylation. Examples of reactive coupling groups can include aldehydes (-COH) and aldehyde reactive linking groups such as hydrazides, alkoxyamines, and primary amines. Examples of reactive coupling groups can include thiol groups (-SH) and sulfhydryl reactive groups such as maleimides, haloacetyls, and pyridyl disulfides. Examples of reactive coupling groups can include photoreactive coupling groups such as aryl azides or diazirines. The coupling reaction may include the use of a catalyst, heat, pH buffers, light, or a combination thereof.

The term "protective group", as used herein, refers to a functional group that can be added to and/or substituted for another desired functional group to protect the desired functional group from certain reaction conditions and selectively removed and/or replaced to deprotect or expose the desired functional group. Protective groups are known to the skilled artisan. Suitable protective groups may include those described in <NPL>). Acid sensitive protective groups include dimethoxytrityl (DMT), tert-butylcarbamate (tBoc) and trifluoroacetyl (tFA). Base sensitive protective groups include <NUM>-fluorenylmethoxycarbonyl (Fmoc), isobutyrl (iBu), benzoyl (Bz) and phenoxyacetyl (pac). Other protective groups include acetamidomethyl, acetyl, tert- amyloxycarbonyl, benzyl, benzyloxycarbonyl, <NUM>-(<NUM>-biphεnylyl)-<NUM>-propy!oxycarbonyl, <NUM>-bromobenzyloxycarbonyl, tert-butyl<NUM> tert-butyloxycarbonyl, l-carbobenzoxamido-<NUM>,<NUM>-trifluoroethyl, <NUM>,<NUM>-dichlorobenzyl, <NUM>-(<NUM>,<NUM>-dimethoxyphenyl)-<NUM>-propyloxycarbonyl, <NUM>,<NUM>-dinitrophenyl, dithiasuccinyl, formyl, <NUM>-methoxybenzenesulfonyl, <NUM>-methoxybenzyl, <NUM>-methylbenzyl, o-nitrophenylsulfenyl, <NUM>-phenyl-<NUM>-propyloxycarbonyl, α-<NUM>,<NUM>,<NUM>-tetramethylbenzyloxycarbonyl, p-toluenesulfonyl, xanthenyl, benzyl ester, N-hydroxysuccinimide ester, p-nitrobenzyl ester, p-nitrophenyl ester, phenyl ester, p-nitrocarbonate, p-nitrobenzylcarbonate, trimethylsilyl and pentachlorophenyl ester.

References herein to a method of therapeutic treatment using the particles of the invention or a formulation containing the particles should be understood as directed to the particles/formulation for use in the method of treatment.

The core of the particles contains one or more hydrophobic polymers (e.g., homopolymer, copolymer, terpolymer, etc.). The polymer may be biodegradable or non-biodegradable. In some embodiments, the core comprises one or more biodegradable polymers.

In general, synthetic polymers are preferred, although natural polymers may be used and have equivalent or even better properties, especially some of the natural biopolymers which degrade by hydrolysis, such as some of the polyhydroxyalkanoates. Representative synthetic polymers are: poly(hydroxy acids) such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid), poly(lactide), poly(glycolide), poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol), polyalkylene oxides such as poly(ethylene oxide), polyalkylene terepthalates such as polyethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly(vinyl chloride), polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene, polyurethanes and co-polymers thereof, derivatized celluloses such as alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, and cellulose sulfate sodium salt (jointly referred to herein as "synthetic celluloses"), polymers of acrylic acid, methacrylic acid or copolymers or derivatives thereof including esters, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as "polyacrylic acids"), poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone), copolymers and blends thereof. Examples of preferred natural polymers include proteins such as albumin, collagen, gelatin and prolamines, for example, zein, and polysaccharides such as alginate, cellulose derivatives and polyhydroxyalkanoates, for example, polyhydroxybutyrate. As used herein, "derivatives" include polymers having substitutions, additions of chemical groups and other modifications routinely made by those skilled in the art.

Examples of preferred non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

In certain embodiments, the hydrophobic polymer is an aliphatic polyester. In preferred embodiments, the hydrophobic polymer is poly(lactic acid), poly(glycolic acid), or poly(lactic acid-co-glycolic acid). The particles are designed to release molecules to be encapsulated or attached over a period of days to weeks. Factors that affect the duration of release include pH of the surrounding medium (higher rate of release at pH <NUM> and below due to acid catalyzed hydrolysis of PLGA) and polymer composition. Aliphatic polyesters differ in hydrophobicity and that in turn affects the degradation rate. The hydrophobic poly(lactic acid) (PLA), more hydrophilic poly(glycolic acid) (PGA) and their copolymers, poly(lactide-co-glycolide) (PLGA) have different release rates. The degradation rate of these polymers, and often the corresponding drug release rate, can vary from days (PGA) to months (PLA) and is easily manipulated by varying the ratio of PLA to PGA. The core can be formed of copolymers including amphiphilic copolymers such as PLGA-PEG or PLURONICS (block copolymers of polyethylene oxide-polypropylene glycol) but this may decrease the benefit of the polyglycerol molecules discussed below.

Other materials may also be incorporated including lipids, fatty acids, and phospholipids. These may be dispersed in or on the particles, or interspersed with the polyglycerol coatings discussed below.

The particles include a shell or coating containing hyperbranched polyglycerol (HPG).

Hyperbranched polyglycerol is a highly branched polyol containing a polyether scaffold. Hyperbranched polyglycerol can be prepared using techniques known in the art. It can be formed from controlled etherification of glycerol via cationic or anionic ring opening multibranching polymerization of glycidol. For example, an initiator having multiple reactive sites is reacted with glycidol in the presence of a base to form hyperbranched polyglycerol (HPG). Suitable initiators include, but are not limited to, polyols, e.g., triols, tetraols, pentaols, or greater and polyamines, e.g., triamines, tetraamines, pentaamines, etc. In one embodiment, the initiator is <NUM>,<NUM>,<NUM>-trihydroxymethyl propane (THP).

A formula for hyperbranched polyglycerol as described in <CIT> is
<CHM>
<CHM>.

The surface properties of HPG can be adjusted based on the chemistry of vicinal diols. For example, the surface properties can be tuned to provide stealth particles, i.e., particles that are not cleared by the MPS due to the presence of the hydroxyl groups; adhesive (sticky) particles, i.e., particles that adhere to the surface of tissues, for example, due to the presence of one or more reactive functional groups, such as aldehydes, amines, oxime, or O-substituted oxime that can be prepared from the vicinal hydroxyl moieties; or targeting by the introduction of one or more targeting moieties which can be conjugated directly or indirectly to the vicinal hydroxyl moieties. Indirectly refers to transformation of the hydroxy groups to reactive functional groups that can react with functional groups on molecules to be attached to the surface, such as active agents and/or targeting moieties, etc. A schematic of this tunability is shown in <FIG> showing bioadhesive polymer.

The hyperbranched nature of the polyglycerol allows for a much higher density of hydroxyl groups, reactive functional groups, and/or targeting moieties than obtained with linear polyethylene glycol. In the present invention, the particles can have a density of surface functionality (e.g., hydroxyl groups) of at least about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> groups/nm<NUM>.

The molecular weight of the HPG can vary. For example, the molecular weight can vary depending on the molecular weight and/or hydrophobicity of the core polymer. The molecular weight of the HPG is generally from about <NUM>,<NUM> to about <NUM>,<NUM>,<NUM> Daltons, from about <NUM>,<NUM> to about <NUM>,<NUM> Daltons, from about <NUM>,<NUM> to about <NUM>,<NUM> Daltons, or from about <NUM>,<NUM> to about <NUM>,<NUM> Daltons. The weight percent of HPG in the copolymer with the core polymer is from about <NUM>% to about <NUM>%, such as about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>%.

Upon self-assembly, particles are formed containing a core containing the hydrophobic polymer and a shell or coating of HPG. HPG coupled to the polymer PLA is shown below:
<CHM>.

The particles of the invention contain one or more therapeutic agents, diagnostic agents, or prophylactic agents encapsulated within the particles. The agents can be covalently or non-covalently associated with the particles.

Molecules can be bound to the hydroxy groups on HPG before or after particle formation. Representative methodologies for conjugating molecules to the hydroxy groups on HPG are described below.

One useful protocol involves the "activation" of hydroxyl groups with carbonyldiimidazole (CDI) in aprotic solvents such as DMSO, acetone, or THF. CDI forms an imidazolyl carbamate complex with the hydroxyl group which may be displaced by binding the free amino group of a ligand such as a protein. The reaction is an N-nucleophilic substitution and results in a stable N-alkylcarbamate linkage of the ligand to the polymer. The "coupling" of the ligand to the "activated" polymer matrix is maximal in the pH range of <NUM>-<NUM> and normally requires at least <NUM> hrs. The resulting ligand-polymer complex is stable and resists hydrolysis for extended periods of time.

Another coupling method involves the use of <NUM>-ethyl-<NUM>-(<NUM>-dimethylaminopropyl) carbodiimide (EDAC) or "water-soluble CDI" in conjunction with N-hydroxylsulfosuccinimide (sulfo NHS) to couple the exposed carboxylic groups of polymers to the free amino groups of ligands in a totally aqueous environment at the physiological pH of <NUM>. Briefly, EDAC and sulfo-NHS form an activated ester with the carboxylic acid groups of the polymer which react with the amine end of a ligand to form a peptide bond. The resulting peptide bond is resistant to hydrolysis. The use of sulfo-NHS in the reaction increases the efficiency of the EDAC coupling by a factor of ten-fold and provides for exceptionally gentle conditions that ensure the viability of the ligand-polymer complex.

By using either of these protocols it is possible to "activate" almost all polymers containing either hydroxyl or carboxyl groups in a suitable solvent system that will not dissolve the polymer matrix.

A useful coupling procedure for attaching ligands with free hydroxyl and carboxyl groups to polymers involves the use of the cross-linking agent, divinylsulfone. This method would be useful for attaching sugars or other hydroxylic compounds with bioadhesive properties to hydroxylic matrices. Briefly, the activation involves the reaction of divinylsulfone to the hydroxyl groups of the polymer, forming the vinylsulfonyl ethyl ether of the polymer. The vinyl groups will couple to alcohols, phenols and even amines. Activation and coupling take place at pH <NUM>. The linkage is stable in the pH range from <NUM>-<NUM>.

Alternatively, the hydroxyl groups can be converted to reactive functional group that can react with a reactive functional group on the molecule to be attached. For example, the hydroxyl groups on HPG can be converted to aldehydes, amines, or O-substituted oximes which can react with reactive functional groups on molecules to be attached. Such transformations can be done before or after particle formation.

Any suitable coupling method known to those skilled in the art for the coupling of ligands and polymers with double bonds, including the use of UV crosslinking, may be used for attachment of molecules to the polymer.

Coupling is preferably by covalent binding but it may also be indirect, for example, through a linker bound to the polymer or through an interaction between two molecules such as strepavidin and biotin. It may also be by electrostatic attraction by dip-coating.

The coupling methods can be done before or after particle formation.

Ultraviolet light filters (such as zinc oxide (ZnO), titanium dioxide (TiO<NUM>), avobenzone, tinosorb S, mexoryl SX, mexoryl XL, helioplex, octinoxate, octocrylene, oxybenzone, octisalate, homosalate, uvinul T <NUM>, cinoxate, aminobenzoic acid, padimate O, ensulizole, dioxybenzone, meradimate, sulisobenzone, trolamine salicylate, enzacamene, bisdisulizole disodium, uvinul A Plus, uvasorb HEB, parsol SLX, amiloxate), antibiotics, antiseptics, antifungals, and anesthetic compounds (such as lidocaine, benzocaine, chloroprocaine, cyclomethycaine, dimethocaine/larocaine, piperocaine, propoxycaine, procaine/novocaine, proparacaine, tetracaine/amethocaine, lidocaine, articaine, bupivacaine, cinchocaine/dibucaine, etidocaine, levobupivacaine, lidocaine/lignocaine, mepivacaine, prilocaine, ropivacaine, trimecaine, lidocaine/prilocaine, lidocaine/tetracaine, prilocaine hydrochloride and epinephrine, lidocaine, bupivacaine, and epinephrine, iontocaine, septocaine, saxitoxin, neosaxitoxin, tetrodotoxin, menthol).

Particularly preferred agents to be delivered include UV filters, such as padimate O (PO), zinc oxide (ZnO), titanium dioxide (TiO<NUM>), silicon oxide coated ZnO or TiO<NUM>, avobenzone, tinosorb S, mexoryl SX, mexoryl XL, helioplex, octinoxate, octocrylene, oxybenzone, octisalate, homosalate, uvinul T <NUM>, cinoxate, aminobenzoic acid, ensulizole, dioxybenzone, meradimate, sulisobenzone, trolamine salicylate, enzacamene, bisdisulizole disodium, uvinul A Plus, uvasorb HEB, parsol SLX, and amiloxate. Incorporated into microparticles, these agents may be used to prevent skin conditions and skin diseases.

Proteins, peptides, carbohydrates, polysaccharides, nucleic acid molecules, and organic molecules, as well as diagnostic agents, can be delivered. The preferred materials to be incorporated are prophylactic agents, such as UV filters, anti-oxidants, anesthetics, corticosteroids, anti-acne agents, and vitamins. Other preferred materials to be incorporated are therapeutic agents. Therapeutic agents include antibiotics, antivirals, anti-parasites (helminths, protozoans), anti-cancer (referred to herein as "chemotherapeutics", including cytotoxic drugs such as doxorubicin, cyclosporine, mitomycin C, cisplatin and carboplatin, BCNU, 5FU, methotrexate, adriamycin, camptothecin, epothilones A-F, and taxol), peptide drugs, anti-inflammatories, and nutraceuticals.

Representative classes of diagnostic materials include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides. Exemplary materials include, but are not limited to, metal oxides, such as iron oxide, metallic particles, such as gold particles, etc. Biomarkers can also be conjugated to the surface for diagnostic applications.

One or more active agents may be formulated alone or with excipients and encapsulated into the microparticles or nanoparticles. Active agents include therapeutic, prophylactic, neutraceutical and diagnostic agents. Any suitable agent may be used. These include organic compounds, inorganic compounds, proteins, polysaccharides, nucleic acids or other materials that can be incorporated using standard techniques.

For imaging, radioactive materials such as Technetium99 (<NUM>Tc) or magnetic materials such as Fe<NUM>O<NUM> could be used. Examples of other materials include gases or gas emitting compounds, which are radioopaque.

Alternatively, the biodegradable polymers may encapsulate cellular materials, such as for example, cellular materials to be delivered to antigen presenting cells as described below to induce immunological responses.

Cell-mediated immunity is needed to detect and destroy virus-infected cells. Most traditional vaccines (e.g. protein-based vaccines) can only induce humoral immunity. DNA-based vaccine represents a unique means to vaccinate against a virus or parasite because a DNA based vaccine can induce both humoral and cell-mediated immunity. In addition, DNA-based vaccines are potentially safer than traditional vaccines. DNA vaccines are relatively more stable and more cost-effective for manufacturing and storage. DNA vaccines consist of two major components - DNA carriers (or delivery vehicles) and DNAs encoding antigens. DNA carriers protect DNA from degradation, and can facilitate DNA entry to specific tissues or cells and expression at an efficient level.

The HPG-coated particles are modified by covalently attaching PEG to the surface. This can be achieved by converting the vicinyl diol groups on the HPG to aldehydes and then reacting the aldehydes with functional groups on PEG, such as aliphatic amines, aromatic amines, hydrazines and thiols. The linker has end groups such as aliphatic amines, aromatic amines, hydrazines, thiols and O-substituted oxyamines. The bond inserted in the linker can be disulfide, orthoester and peptides sensitive to proteases.

PEG with a functional group or a linker can form a bond with aldehyde on PLA-HPGALD and reverse the bioadhesive (sticky) state of PLA-HPGALD to stealth state. This bond or the linker is labile to pH change or high concentration of peptides, proteins and other biomolecules. After administration systematically or locally, the bond attaching the PEG to PLA-HPGALD can be reversed or cleaved to release the PEG in response to the environment and expose the PLA-HPGALD particles to the environment. Subsequently, the particles will interact with the tissue and attach the particles to the tissues or extracellular materials such as proteins. The environment can be acidic environment in tumors, reducing environment in tumors, or protein rich environment in tissues.

Methods of making polymeric particles are known in the art. Common microencapsulation techniques include, but are not limited to, spray drying, interfacial polymerization, hot melt encapsulation, phase separation encapsulation (spontaneous emulsion microencapsulation, solvent evaporation microencapsulation, and solvent removal microencapsulation), coacervation, low temperature microsphere formation, and phase inversion nanoencapsulation (PIN). A brief summary of these methods is presented below.

In some embodiments, the particles are prepared using an emulsion-based technique. In particular embodiments, the particles are prepared using a double emulsion solvent evaporation technique. For example, the amphiphilic material and the hydrophobic cationic material are dissolved in a suitable organic solvent, such as methylene chloride or dichloromethane (DCM), with or without agent to be encapsulated.

In phase separation microencapsulation techniques, a polymer solution is stirred, optionally in the presence of one or more active agents to be encapsulated. While continuing to uniformly suspend the material through stirring, a nonsolvent for the polymer is slowly added to the solution to decrease the polymer's solubility. Depending on the solubility of the polymer in the solvent and nonsolvent, the polymer either precipitates or phase separates into a polymer rich and a polymer poor phase. Under proper conditions, the polymer in the polymer rich phase will migrate to the interface with the continuous phase, encapsulating the active agent(s) in a droplet with an outer polymer shell.

Spontaneous emulsification involves solidifying emulsified liquid polymer droplets formed above by changing temperature, evaporating solvent, or adding chemical cross-linking agents. The physical and chemical properties of the encapsulant, as well as the properties of the one or more active agents optionally incorporated into the nascent particles, dictates suitable methods of encapsulation. Factors such as hydrophobicity, molecular weight, chemical stability, and thermal stability affect encapsulation.

Methods for forming microspheres using solvent evaporation techniques are described in<NPL>); <NPL>); <NPL>); <NPL>); and <CIT>. The polymer is dissolved in a volatile organic solvent, such as methylene chloride. One or more active agents to be incorporated are optionally added to the solution, and the mixture is suspended in an aqueous solution that contains a surface active agent such as poly(vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporated, leaving solid microparticles/nanoparticles. This method is useful for relatively stable polymers like polyesters and polystyrene.

Nanoparticles can also be formed using the phase inversion nanoencapsulation (PIN) method, wherein a polymer is dissolved in a "good" solvent, fine particles of a substance to be incorporated, such as a drug, are mixed or dissolved in the polymer solution, and the mixture is poured into a strong non solvent for the polymer, to spontaneously produce, under favorable conditions, polymeric microspheres, wherein the polymer is either coated with the particles or the particles are dispersed in the polymer. See, e.g., <CIT> The method can be used to produce monodisperse populations of nanoparticles and microparticles in a wide range of sizes, including, for example, about <NUM> nanometers to about <NUM> microns.

Nanoparticles can be prepared using microfluidic devices. A polymeric material is mixed with a drug or drug combinations in a water miscible organic solvent. The water miscible organic solvent can be one or more of the following: acetone, ethanol, methanol, isopropyl alcohol, acetonitrile and Dimethyl sulfoxide (DMSO). The resulting mixture solution is then added to an aqueous solution to yield nanoparticle solution.

Hyperbranched polyglycerol (HPG) can be covalently bound to one or more polymers that form the core of the particles using methodologies known in the art. For example, HPG can be covalently coupled to a polymer having carboxylic acid groups, such as PLA, PGA, or PLGA using DIC/DMAP. In another example, the HPG can be initiated from special functionalized initiators to facilitate the conjugation to more materials. These special initiators include disulfide (<NPL>)).

Certain properties of the PLA-HPG conjugate are important for the observed effects. Because high molecular weight HPG has better resistance of non-specific adsorption to biomolecules, the low molecular weight components are removed from the synthesized HPG by multiple solvent precipitations and dialysis.

In the preferred embodiment, a polyhydroxy acid such as PLA is selected as the hydrophobic core material because it is biodegradable, has a long history of clinical use, and is the major component of a NP system that is advancing in clinical trials. To covalently attach the PLA to HPG, the previous approach was to first functionalize the HPG with an amine and then conjugate the carboxylic group on PLA to the amine. This approach is efficient but cannot be used to make HPG as surface coatings since any amines that do not react with PLA will lead to a net positive charge on the neutral HPG surface and reduce the ability of HPG to resist adsorption of other molecules on the surface. To avoid this, the approach in the examples uses a one-step esterification between PLA and HPG, which maintained the charge neutral state of the HPG.

The particles may have any zeta potential. The particles can have a zeta potential from -<NUM> mV to + <NUM> mV, -<NUM> mV to +<NUM> mV, from -<NUM> mV to +<NUM> mV, from -<NUM> mV to +<NUM> mV, from -<NUM> mV to +<NUM> mV, from -<NUM> mV to +<NUM> mV, from -<NUM> mV to +10mV, or from -5mV to +<NUM> mV. The particles can have a negative or positive zeta potential. In some embodiments the particles have a substantially neutral zeta potential, i.e. the zeta potential is approximately <NUM> mV. In preferred embodiments the particles have a zeta potential of approximately -<NUM> to about <NUM> mV, preferably from about -<NUM> to about <NUM> mV, more preferably from about -<NUM> to about <NUM> mV.

The particles may have any diameter. The particles can have a diameter of about <NUM> to about <NUM> microns, about <NUM> to about <NUM> microns, about <NUM> to about <NUM> microns, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, or about <NUM> to about <NUM>. In preferred embodiments, the particle is a nanoparticle having a diameter from about <NUM> to about <NUM>. In more preferred embodiments, the particles are nanoparticles having a diameter from about <NUM> to about <NUM>, preferably from about <NUM> to about <NUM>.

The polydispersity is from about <NUM> to <NUM>, preferably from about <NUM> to about <NUM>, more preferably from about <NUM> to about <NUM>, more preferably from about <NUM> to about <NUM>, most preferably from about <NUM> to about <NUM>.

The particles can be formulated with appropriate pharmaceutically acceptable carriers into pharmaceutical compositions for administration to an individual in need thereof. The formulations can be administered topically (e.g., to the skin via non-invasive topical application). Other routes of administration include, but are not limited to, transdermal.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.

Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(<NUM>-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-<NUM>-oleate, sorbitan acylate, sucrose acylate, PEG-<NUM> laurate, PEG-<NUM> monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-<NUM> cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, POLOXAMER® <NUM>, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-beta-alanine, sodium N-lauryl-beta-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, benzalkonium, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).

The formulation is typically buffered to a pH of <NUM>-<NUM>. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

Water-soluble polymers may be used in formulations. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

Carrier also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. Formulations can be prepared using one or more topical or pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).

The particles can be used for a variety of applications including protecting tissue from UV light, drug delivery, tissue engineering, etc. Stealth properties can be useful for topical delivery, for example, diffusion into hair follicles to release agents to promote hair growth/prevent hair loss or agents that promote hair loss.

The particles can be used to deliver one or more therapeutic agents, diagnostic agents, prophylactic agents, nutraceuticals, or combinations thereof to the skin. As discussed above, the PEG can be cleaved to make the particles adhere or stick to tissue or other biological materials, such as skin (e.g., sticky particles). Incubation of HPG-coated particles with the reagent NaIO<NUM>, which converts vicinyl diol groups to aldehyde groups, showed an estimated <NUM> aldehyde groups/nm<NUM> after about <NUM> minutes. Surface immobilization of HPG particles on lysine-coated slides increased as the incubation time with NaIO<NUM> increased.

Aldehyde-functionalized HPG-coated particles loaded with the dye DiD were incubated with pig skin for <NUM> hours. The relative fluorescence of the aldehyde-functionalized particles was three-fold higher than the relative fluorescence of HPG-coated particles.

In view of the data above, the particles can be used for delivery of one or more active agents to the skin. In some embodiments, the particles can be used as or formulated in a sunscreen. For example, the particles were loaded with padimate O (PO), an organic compound used in suncreens. Under conditions mimicking the pH range of human sweat (<NUM> and <NUM>), the particles retained more than about <NUM>% of the PO after <NUM> hours. The particles also exhibited improved UV absorption compared to PO dispersed in water or aqueous buffer.

Other agents include agents for treating skin aging, such as anti-reactive oxygen species (ROS) therapies.

The particles can also be used to deliver agents to hair follicles, for examples, agents to promote hair growth or reduce hair loss or therapies to remove hair. The stealth nature of the particles can allow diffusion of the particles into the hair follicles.

A chronic wound is a wound that does not heal normally. Wounds that do not heal within three months are often considered chronic. One embodiment provides administering an effective amount of the particles to a chronic wound to promote or enhance healing.

The disclosed particles can also be used to treat fibrotic wounds. Fibrotic wounds have dysregulated healing and typically delayed healing. Fibrosis can be defined as the replacement of the normal structural elements of the tissue by distorted, non-functional and excessive accumulation of scar tissue. One embodiment provides a method for treating fibrotic wounds by administering an effective amount of the disclosed particles to promote or enhance fibrotic wound healing.

The particles can also be used with wound dressings. In one example, a wound dressing has a layer of particles thereon. The layer of particles is configured to come into contact with the wound when the wound dressing is applied to a wound. The particles can be impregnated in the wound dressing or coated on the wound dressing using conventional techniques. The wound dressing can be made of absorbent materials such as cotton or fleece. The wound dressing can also be made of synthetic fibers, for example, polyamide fibers. In certain examples, the wound dressing can have multiple layers including an adhesive layer, an absorbent layer, and moisture regulation layer. The wound dressing can also include antimicrobial agents, antifungal agents, and other active agents to promote wound healing such as cytokines and growth factors discussed above.

Another embodiment provides a method for treating a wound by administering an effective amount of the disclosed particles to the wound to promote or induce hemostasis and then applying a wound dressing to the wound.

The advantage of these particles is that they can be converted so that they adhere to the skin and/or applied material, where they are retained at the site of injury to provide sustained treatment. Mixtures releasing different amounts or different drugs at different times are particularly advantageous for treatment of wounds such as diabetic wound ulcers. Ligands can be selected to enhance the particles being retained at the site, by binding to extracellular matrix or through non-specific electrostatic binding.

The present invention will be further understood in view of the following nonlimiting examples.

Polylactic acid (Mw = <NUM> kDa, Mn = <NUM>. 4kDa) was obtained from Lactel.

H<NUM>N-PEG(<NUM>)-OCH<NUM> was obtained from Laysan.

Anhydrous dimethylformide, dichloromethane, diisopropylcarboimide, dimethylaminopyridie, potassium methoxide, camptothecin, polyvinyl alcohol, paraformaldehyde, TWEEN® <NUM>, and <NUM>,<NUM>,<NUM>-trihydroxymethyl propane were obtained from the Sigma-Aldrich.

Anhydrous dry ether, methanol, acetonitrile and dimethylsulfoxide were obtained from J.

<NUM>,<NUM>'-Dioctadecyl-<NUM>,<NUM>,<NUM>',<NUM>'-Tetramethylindodicarbocyanine,<NUM> Chlorobenzenesulfonate Salt (DiD) was obtained from Invitrogen.

Super frost microscope slides were obtained from Thermo Scientific.

Microdialysis tubing was from Thermo Scientific.

IR-<NUM> iodide, hydroxylamine solution (<NUM>%), glycerol, polyvinyl alcohol, NaIO4 and bovine serum albumin (BSA) were obtained from the Sigma-Aldrich.

The <NUM>,<NUM>'-Dioctadecyl-<NUM>,<NUM>,<NUM>',<NUM>'-Tetramethylindodicarbocyanine,<NUM> Chlorobenzenesulfonate Salt (DiD) and DAPI stain were ordered from Invitrogen.

Donkey normal serum and Rabbit-anti-CD31 antibody was provided by Abcam and the Donkey-anti-rabbit secondary antibody tagged with Alexa488 fluorophore was from Invitrogen.

Aldehyde Quantification Assay Kit (Fluorometric) and were from Abcam.

Sunscreen lotion (SPF <NUM>) was purchased from Walgreens.

Sunscreen oil (SPF <NUM>) was from L'Oréal Paris.

Hyperbranched polyglycerol (HPG) was synthesized by anionic polymerization. Briefly, <NUM> mmol <NUM>,<NUM>,<NUM>-trihydroxypropane (THP) was added into an argon protected flask in a <NUM> oil bath and <NUM> mmol KOCH<NUM> was added. The system was hooked up to a vacuum pump and left under vacuum for <NUM>. The system was refilled with argon and <NUM> glycidol was added by a syringe pump over <NUM> hours. The HPG was dissolved in methanol and precipitated by addition of acetone. HPG was purified <NUM>-<NUM> times with methanol/acetone precipitation. To further remove the low molecular weight HPG, <NUM>-<NUM> HPG was placed in a <NUM> dialysis tube (<NUM>-<NUM> cut-off) and dialyzed against deionized (DI) water. The water was replaced two times every <NUM> hours. HPG was precipitated with acetone and then dried under vacuum at <NUM> for <NUM>.

PLA (<NUM>) and <NUM> HPG were dissolved in dimethyl formamide (DMF) and dried over molecular sieves overnight. <NUM> diisopropylcarboimide (DIC) and <NUM> <NUM>-(N,N-dimethylamino)pyridine (DMAP) were added and the reaction proceeded for <NUM> days at room temperature under stirring. The product was precipitated by pouring the reaction into cold diethyl ether (ether) and collecting the precipitate by centrifugation. The product was redissolved in dichloromethane (DCM) and precipitated again with a cold mixture of ether and methanol. The product was washed with a cold mixture of ether and methanol. The polymer was dried under vacuum for <NUM> days.

To synthesize PLA-PEG, <NUM> PLA and <NUM> MPEG-NH<NUM> were dissolved in DMF and dried over molecular sieves overnight. <NUM> DIC was added and the reaction proceeded for <NUM> days at room temperature under stirring. The product was precipitated by pouring the reaction into cold ether and collecting the precipitate by centrifugation. The product was redissolved in DCM and precipitated again with cold ether, washed with a cold mixture of ether and methanol and dried under vacuum for <NUM> days.

Fifty mg of PLA-HPG copolymer dissolved in <NUM>-<NUM> of ethyl acetate/dimethyl sulfoxide (DMSO) (<NUM>: <NUM>) was added to <NUM> DI water under vortexing and subjected to probe sonication for <NUM> cycles at <NUM> sec each. The resulting emulsion was diluted in <NUM> DI water under stirring. It was stirred for at least <NUM> hours or attached to a ratovapor to evaporate the ethyl acetate and then applied to an Amico ultra centrifuge filtration unit (<NUM> cut-off). The NPs were washed by filtration <NUM> times then suspended in a <NUM>% sucrose solution. The NPs were kept frozen at -<NUM>.

The PLA-PEG NPs were made using a single emulsion technique. <NUM> PLA-PEG copolymer dissolved in <NUM>-<NUM> ethyl acetate/DMSO (<NUM>:<NUM>) was added to <NUM> DI water with <NUM>% PVA under vortexing and subjected to probe sonication for <NUM> cycles of <NUM> sec each. The resulting emulsion was diluted in <NUM> DI water with <NUM>% Tween® <NUM> with stirring. The emulsion was stirred for at least <NUM> hours or attached to a ratovapor to evaporate the ethyl acetate and then the solution was applied to an Amico ultra centrifuge filtration unit (<NUM> cut-off). The NPs were washed by filtration for <NUM> times then suspended in a <NUM>% sucrose solution.

The NPs were characterized with TEM. A drop of nanoparticle suspension was applied on the top of carbon coated copper grids and most of the droplet was removed with a piece of filter paper. The thin layer of NPs suspension was dried for <NUM>-<NUM> and then a droplet of uranyl acetate was applied. Most of the droplet was removed with a filter paper and left to dry for <NUM>. The sample was mounted for imaging with TEM. The size distribution of NPs was analyzed in Image J. The hydrodynamic size of NPs was determined by dynamic laser scattering (DLS). NPs suspension was diluted with DI water to <NUM>/ml and <NUM> was loaded into the cell for detection.

To determine the concentration of the dye in NPs, <NUM>µL DMSO was added to <NUM>µL NPs in aqueous solution. The solution was vortexed and left in the dark for <NUM>. The concentration of the dye was quantified with a plate reader by fluorescence of the DiD dye at <NUM> with an excitation wavelength at <NUM>.

<NUM>H NMR spectra for HPG and PLA-HPG block-copolymer were recorded on a <NUM> Agilent instrument using DMSO-d6 as solvent. Inverse gated <NUM>C NMR spectra for HPG were recorded on a <NUM> Agilent instrument with methanol-d4 as solvent.

The DPn (number-average degree of polymerization) for HPG was calculated according to the inverse gated <NUM>C NMR spectra for HPG with the following equation: <MAT>.

The functionality of the core molecule (TMP), fc, is <NUM>.

The Mn of HPG is calculated with the following equation:<MAT>.

Both particles have a biodegradable PLA core, which can be used to load hydrophobic agents, and a hydrophilic shell of HPG or PEG. HPG was made by anionic polymerization and characterized by <NUM>H NMR and <NUM>C NMR. PLA-HPG copolymer was synthesized by esterification and the conjugation of PLA-HPG was confirmed by <NUM>H NMR. The weight percentage of HPG in PLA-HPG was about <NUM>% as calculated from the NMR results.

PLA-HPG NPs were made from a single emulsion as described above. PLA-PEG copolymer was synthesized by the conjugation of PLA-COOH with amine terminated mPEG and also characterized with <NUM>H NMR. The weight percentage of PEG was about <NUM>% as calculated from the NMR results.

Transmission electronic microscopy (TEM) confirmed the spherical shape of the PLA-HPG and PLA-PEG NPs. The hydrodynamic diameter of NPs was <NUM> as measured by dynamic light scattering (DLS) (Table <NUM>).

Microdialysis tubes were filled with <NUM>µL of NPs loaded with <NUM>,<NUM>'-dioctadecyl-<NUM>,<NUM>,<NUM>',<NUM>'-tetramethylindodicarbocyanine, <NUM>-chlorobenzenesulfonate Salt (DiD) and placed on a floater in a large beaker with <NUM> PBS at <NUM>. Tubes were removed in triplicates at different time points. The PBS was changed every <NUM> hours. The dye left in the dialysis tube was quantified by fluorescence.

DiD-loaded NPs release a minimal amount of dye (~<NUM>%) over <NUM> days of continuous incubation in PBS (<FIG>). Both PLA-HPG and PLA-PEG NPs were loaded with equivalent amounts of DiD.

PLA-HPG NPs (<NUM>/ml) in a <NUM>-well plate (small vial) were incubated with <NUM> NaIO<NUM> and at each time point, the reactions were quenched with <NUM> Na<NUM>SO<NUM>. The NPs were washed two times with DI water in an AcroPrep filter plate with <NUM> cut-off (or amicon ultra filter <NUM> with <NUM> cut-off) and then suspended in DI water.

The aldehydes on NPs were quantified with an aldehyde quantification assay kit (Abcam). The PLA-HPG NPs were used as a background subtraction control. The amount of aldehyde was calculated by comparing to a reference curve. The reference curve was made by using the aldehyde standard provided with the kit. The amount of aldehyde on each particle was calculated based on <NUM> hydrodynamic diameter of NPs and an assumed NP density of <NUM>/cm<NUM>. For microarray printing, NPs load with DiD dye were suspended in PBS buffer containing <NUM>% glycerol and <NUM>% triton-X100 at a concentration of <NUM>/ml in a <NUM>-well plate. The NPs were arrayed on lysine coated slides using a Spotbot microrrayer from Arrayit. After <NUM> hour incubation in a humidity chamber, the printed slides were washed extensively with PBS <NUM> times, <NUM> each. After a quick rinse with DI water, the slides were blow-dried with argon and subjected for imaging.

For ligand or protein attachment, in a <NUM>-well plate (or small vials), PLA-HPGALD NPs were incubated with ligands or proteins (NaCNBH<NUM> should be added for proteins or ligands modified with amines or hydrazines) for <NUM>-<NUM> hours and the reaction was quenched with an excess amount of hydroxylamine (or ethanolamine for proteins or ligands modified with amines or hydrazines) solution in TRIS buffer (PH=<NUM>). The NPs were transferred to an AcroPrep filter plate with <NUM> cut-off (or amicon ultra filter <NUM> with <NUM> cut-off or gel filtration for proteins and other large molecules) and washed two times with DI water or buffer.

To reduce the PLA-HPGALD NPs (sticky, also referred to herein as bioadhesive nanoparticles, BNPs) back to PLA-HPG NPs (also referred to herein as non-bioadhesive nanoparticles, NNPs), PLA-HPGALD NPs were incubated with NaBH<NUM> in NaH<NUM>PO<NUM> (<NUM>, PH=<NUM>) and the reaction was quenched with acetic acid and neutralized with PBS buffer. The NPs were washed with DI water twice. The blood circulation experiments were performed to test the stealth properties of the nanoparticles.

PLA-HPGALD NPs (BNPs) could be reversed to PLA-HPGReversed (stealth) NPs by NaBH<NUM> treatment, though one alcohol group is lost with the reduction-reversal cycle since each vicinal diol on HPG is oxidized by NaIO<NUM> to an aldehyde and each aldehyde is reduced to a single alcohol by NaBH<NUM>. The blood circulation confirmed that the PLA-HPGALD NPs lost almost all their stickiness after treatment with NaBH<NUM>. The back and forth tunability also demonstrated the robustness of the HPG coating on the nanoparticles.

Polylysine coated glass slides were used as a tissue mimic to evaluate the bioadhesive property of PLA-HPGALD NPs (BNPs). PLA-HPGALD NPs with different concentrations of aldehydes were prepared using a high-throughput procedure, where regular <NUM>-well plates and <NUM>-well filter plates were used to prepare the NPs and printed onto polylysine coated slides with a microarrayer. The PLA-HPG NPs (NNPs) without NaIO<NUM> treatment did not adhere to glass slides and only background signal was detected. However, by transforming the surface property with NaIO<NUM>, the amount of NPs immobilized on the glass slide increased as a function of duration of NaIO<NUM> treatment, indicating that the bioadhesive property of the PLA-HPG NPs can be tuned by control of NaIO<NUM> treatment.

For microarray printing, NPs load with DiD dye were suspended in PBS buffer containing <NUM>% glycerol and <NUM>% TRITON®-X100 at a concentration of <NUM>/ml in a <NUM>-well plate. The NPs were arrayed on lysine coated slides using a SPOTBOT® microrrayer from ARRAYIT®. After <NUM> hour incubation in a humidity chamber, the printed slides were washed extensively with PBS <NUM> times, <NUM> each. After a quick rinse with DI water, the slides were blow-dried with argon and subjected for imaging.

The bioadhesive property of PLA-HPG NPs on tissues was evaluated by applying suspended NPs ex vivo to the external surface of pig skin. Fresh pig skin was obtained from a local slaughterhouse and the hair was carefully removed by a trimmer, making sure no damage occurred to the skin. The skin was frozen at -<NUM>. The skin was thawed on ice before use. Thawed pig skin was washed with PBS buffer and cut to 2x2 cm pieces. DiD-loaded PLA-HPG NPs (NNPs) and PLA-HPGALD NPs (BNPs) in PBS were topically applied to pig skin and incubated for <NUM> in a humidity chamber at <NUM>. After incubation, skin was washed with plenty of PBS buffer and frozen in OCT. The frozen skin was sectioned into <NUM>-<NUM> slices, mounted on glass slides, and imaged with an EVOS fluorescence microscope.

For the live imaging study of adherence of PLA-HPGALD NPs to the skin on Nude mice, the dorsal skin of each Nude mouse was cleaned with an alcohol pad and <NUM>/ml of IR-<NUM>/PLA-HPGALD NPs (<NUM>%) in PBS was applied to the skin. The nanoparticles remaining on the skin were imaged by XENOGEN®. The mice were housed individually and imaged at each time point. For evaluation of PLA-HPGALD NPs water resistance and mechanical removal, one group of Nude mice (n=<NUM>) was wiped with a wet towel and the other group of mice was washed with water. The mice were subsequently dried with kimwipes and sent for live imaging.

The results are shown in <FIG>. PLA-HPGALD NPs showed greater retention on pig skin than PLA-HPG NPs (P<<NUM>). The fluorescence intensity was quantified from the fluorescence images.

Because the HPG coating is rich in vicinal diols, PLA-HPG NPs can be readily oxidized to aldehyde-terminated PLA-HPGALD NPs by sodium periodate (NaIO4) treatment. This was validated by H<NUM>NMR and Schiff's agent analysis. The surface density of aldehydes on PLA-HPGALD NPs was monitored as a function of incubation time with NaIO<NUM> and it reached its saturation at about <NUM> (<FIG>). The final surface density of aldehydes on PLA-HPGALD NPs approached <NUM>/nm<NUM> (<NUM> aldehydes/PLA molecule), indicating that the majority of surface vicinal diols were converted to aldehydes. This surface density of functional groups is at least one order of magnitude higher than previously reported on biodegradable NPs (<NPL>)). Moreover, the surface density of the aldehydes can be controlled by incubation time with NaIO<NUM>. No detrimental effects of aldehyde conversion were observed on NPs by TEM imaging. The average diameter of NPs was approximately <NUM> by dynamic light scattering (DLS) measurement (Table <NUM>).

The bioadhesive properties of the PLA-HPGALD NPs (BNPs) using polylysine coated glass slides as a tissue mimic (<NPL>)) was investigated. PLA-HPGALD NPs (BNPs) with different concentrations of aldehyde were prepared and printed onto polylysine coated slides with a microarrayer. PLA-HPG NPs (NNPs) did not adhere to glass slides (<FIG>). However, after oxidizing surface HPG vicinal diols into aldehydes with NaIO<NUM>, the amount of PLA-HPGALD NPs immobilized on the glass slide increased as a function of NaIO<NUM> treatment duration (<FIG>), indicating that the bioadhesive property of the PLA-HPGALD NPs increases with a longer duration of NaIO<NUM> treatment. Moreover, the large capacity for surface aldehyde modification allows for tuning adhesiveness for specific topical applications.

Delivery vehicles for UV-filters should ideally remain only on the skin surface, without penetration into the epidermis, dermis, or hair follicles, in order to avoid potential health risks (<NPL>)). Thus, the retention and the penetration of PLA-HPGALD NPs ex vivo to PLA-HPG NPs by applying suspended particles topically onto pig skin was compared. To facilitate imaging and quantification, both NPs were loaded with a hydrophobic dye, <NUM>%<NUM>,<NUM>'-dioctadecyl-<NUM>,<NUM>,<NUM>',<NUM>' -tetramethylindodicarbocyanine,<NUM>-chlorobenzenesulfonate salt (DiD) (<NPL>)). Both the DiD/PLA-HPG NPs and DiD/PLA-HPGALD NPs were characterized by TEM and DLS (Table <NUM>), and had a similar spherical morphology. After incubation for <NUM> hours with both NPs, followed by extensive washing, PLA-HPGALD NPs showed substantially higher retention on pig skin compared to NNPs (<FIG>). No penetration of PLA-HPGALD NPs was observed on any pig skin samples; however, PLA-HPG NPs penetrated into the pig skin follicles without significant retention on the stratum corneum. Pig skin is considered a good mimic for human skin in a variety of applications including penetration studies for chemicals and nanoparticles (<NPL>);<NPL>)). These results indicate that PLA-HPGALD NPs exhibit no skin penetration whereas the PLA-HPG NPs exhibit considerable penetration into follicles, reflect the adhesion of PLA-HPGALD NPs to proteins on the skin surface, which prevents diffusion of nanoparticles to deeper skin layer or into follicles.

The water resistance and potential for removal of PLA-HPGALD NPs by encapsulating an infrared dye, IR-<NUM>, into BNPs (<NUM>% loading), and measuring nanoparticle skin concentrations with in vivo imaging was investigated. The IR-<NUM>/PLA-HPGALD NPs were characterized by TEM and DLS (Table <NUM>). After extensive washing with water, no significant change in fluorescence was observed; however, the PLA-HPGALD NPs were removed after wiping with a wet towel (<FIG>). If untreated, PLA-HPGALD NPs concentration diminished markedly (approximately <NUM>%) within <NUM> hr and disappearance was essentially complete after five days (Figure 5A).

These examples show that PLA-HPGALD NPs will interact with tissues since the bioadhesive property of PLA-HPGALD NPs is resulted from the Schiff-base bond between the aldehyde groups on PLA-HPGALD NPs and the amine groups in tissue surface.

These results support the use of PLA-HPG NPs in local delivery where an extended retention at delivery sites is needed. The density of the aldehydes on NPs can be controlled thereby providing tunability in the behavior of the PLA-HPGALD NPs for local delivery, especially since the PLA-HPG NPs penetrated into hair follicles on the pig skin.

Sunscreens based on PLA-HPGALD NPs can simplify the current sunscreen formulation as well as eliminate the use of irritants and/or allergens. PLA-HPGALD NPs are ideal vehicles for sunscreen application since they are water-soluble but their interaction with skin is water-resistant. The PLA-HPGALD NPs disappear from skin naturally by exfoliation of the stratum corneum; removal can be accelerated mechanically by towel drying. Moreover, nanoparticles, of the size used in this study, yield more transparent suspensions, which may be favored in topical applications for aesthetic reasons.

PLA-HPG polymer and PO (an organic compound used in sunscreens), in certain ratio (ratio from <NUM>:<NUM> to <NUM>:<NUM> and total mass of <NUM>-<NUM>), were dissolved in <NUM>-<NUM> solvent mixture (Ethyl acetate:DMSO = <NUM>:<NUM>) was added into <NUM> DI water under vortexing and then subjected to probe sonication for <NUM> cycles at <NUM> sec each. The resulting emulsion was diluted in <NUM> DI water with stirring. It was hooked up to a rotovapor to evaporate the ethyl acetate and then applied to an Amico ultra centrifuge filtration unit (<NUM> cut-off). The PO/PLA-HPG NPs were washed by filtration <NUM> times then suspended in DI water. The same procedure was implemented to produce PLA-HPGALD NPs, as PO/PLA-HPGALD NPs can be oxidized from PO/PLA-HPG NPs nanoparticles.

To quantify the PO loading, the nanoparticles were dissolved in DMSO and the UV absorbance at <NUM> was measured with a plate reader. The amount of PO was calculated by comparing to a reference curve.

All PO/PLA-HPGALD NPs contained <NUM>% PO. The spherical shape of the POBNPs was confirmed by TEM. A hydrodynamic diameter of <NUM> for PO/PLA-HPGALD NPs was measured by DLS. The results for hydrodynamic diameter of all the NPs generated in these studies are presented in Table <NUM>.

The stability of PO encapsulation in NPs was evaluated by measuring the release of PO in buffer mimicking the pH range (<NUM>-<NUM>) of human sweat.

To quantify PO release from PLA-HPGALD NPs, a suspension of <NUM> NPs loaded with PO in a dialysis tube (<NUM> cut-off) was dialyzed against <NUM> PBS with <NUM>% SDS at <NUM>. At each time point, <NUM>µL solution was removed and <NUM>µL PBS with <NUM>% SDS was added. The amount of released PO was quantified by UV adsorption at <NUM> with a plate reader.

The results are shown in <FIG>. Under conditions mimicking the pH range of human sweat (<NUM> and <NUM>), PLA-HPGALD NPs loaded with PO retained more than about <NUM>% of the PO after <NUM> hours. The particles also exhibited improved UV absorption compared to PO dispersed in water or aqueous buffer.

Most organic UV filters prevent sunburn by absorbing UV radiation. Therefore, their effectiveness can be estimated by measuring their UV absorption efficiency. Photoinduced changes in UV filters often produce toxic intermediates including ROS that are destructive to multiple cellular components including gDNA (<NPL>)). It has been reported that encapsulating UV filters in polymeric nanoparticles improves filter photostability and delays photodegradation of the UV filters (<NPL>)). To test whether encapsulating UV-filters in PLA-HPGALD NPs would in turn confine any generated ROS within the nanoparticles, thereby eliminating potential side-effects, the PO/PLA-HPGALD NPs and PO suspension were mixed with DHR and exposed to UV.

The UV absorption of PO/PLA-HPGALD NPs was evaluated by measuring their absorption spectrum within the UV range (<NUM>-<NUM>).

PO/PLA-HPGALD NPs suspended in water, PO emulsified in water and PO dissolved in mineral oil at a PO concentration of <NUM>/ml were aliquoted into a UV transparent plate and scanned through the UV absorbance spectrum from <NUM>-<NUM> with a plate reader. Blank PLA-HPGALD NPs, water and mineral oil were also scanned as background controls. The PO emulsion in water was made by probe sonication. For the DHR assay, PO/PLA-HPGALD NPs, PLA-HPGALD NPs, PO water emulsion at a PO concentration of <NUM>/ml was incubated with DHR in <NUM> well plate. After exposing to UV-B (<NUM>-<NUM>), plate fluorescence was read at Ex/Em <NUM>/<NUM>.

Dihydrarhodamine (DHR), a widely used ROS probe (<NPL>)), was used to detect reactive radicals generated by PO after UV exposure. DHR was mixed with PO/PLA-HPGALD NPs, emulsified PO, and PLA-HPGALD NPs separately and exposed to UV. DHR in PBS was used as a control because it absorbs UV at <NUM>-<NUM> and becomes fluorescent.

PO/PLA-HPGALD NPs were compared to PO emulsified in polyvinyl alcohol (PVA) solution (PO/PVA), PO in mineral oil (PO/oil), sunscreen (L'Oreal sunscreen oil spray) in mineral oil (sunscreen/oil); blank PLA-HPGALD NPs, mineral oil and PVA solution were used as controls (<FIG>). All solutions contained equivalent PO concentrations (<NUM>/ml). PO/PLA-HPGALD NPs showed a <NUM>-fold higher absorption compared to the PO emulsion in PVA solution and sunscreen diluted in mineral oil (active ingredients adjusted to <NUM>/ml) after background subtraction of the appropriate base material (<FIG>).

The PO/PVA emulsion is a simplified, representative version of a sunscreen formulation (<NPL>)); most current sunscreens are based on an emulsion of UV filters (<NPL>)). The sunscreen oil used in this example is an oil spray with the same active ingredients and SPF value as the sunscreen lotion used in the animal studies. These results indicate a significant improvement in UV absorption efficiency of PO/PLA-HPGALD NPs compared to PO dissolved in mineral oil.

The PLA-HPGALD NPs had a negligible effect on the background fluorescence of DHR as measured by the control since they did not absorb UV. The fluorescence from the PO suspension is much higher than the control (<FIG>). It is believed that the free ROS generated from the photoactivated PO after UV exposure oxidized the DHR into fluorescent species. In contrast, by confining the ROS within NPs, the PO/PLA-HPGALD NPs significantly decreased the background fluorescence.

The protective effect of the PO/PLA-HPGALD NPs against sunburn on the dorsal skin of Nude mice was evaluated.

Nude mice were anesthetized with Ketamine/Xylazine, and their dorsal skin was cleaned with <NUM>% alcohol and demarcated into four quadrants. One quadrant was used as a PBS control and other areas were treated with sunscreen, PO/PLA-HPGALD NPs or blank PLA-HPGALD NPs. Only the dorsal epidermis was exposed to the UV lamp (UV-A and UV-B, <NUM>-<NUM>, 8W) for one min (2160J/m<NUM>) and the remaining skin was covered with screens. The mice were left in separate cages and monitored until they woke up. Three days after UV exposure, the dorsal skin was removed and prepared for histology. Images were analyzed for epidermal thickness and keratin content using Imaged.

Three days after UV exposure, skin treated with both PO/PLA-HPGALD NPs and sunscreen contained no visible erythema, edema or ulceration. However, both skin patches treated with PBS and blank PLA-HPGALD NPs were damaged considerably by the same UV exposure. A similar pattern of UV toxicity was seen after staining the dorsal skin with hematoxylin and eosin (H&E) (<FIG>). There was significant acanthosis with prominent rete ridges present in the unprotected samples, consistent with epidermal hypertrophy, whereas the skin protected by sunscreen or PO/PLA-HPGALD NPs appeared comparable to normal controls. The UV filter (PO) concentration in PO/PLA-HPGALD NPs was less than <NUM>% of that contained in the sunscreen, yet the PO/PLA-HPGALD NPs achieved a similar gross UV protection effect. Trichome staining was also employed to measure the anti-UV effect against sunburn (<FIG>). The skin protected by sunscreen showed thickened orthokeratosis, a more subtle epidermal response than to UV-damage, relative to the skin protected by PO/PLA-HPGALD NPs and the normal skin control. Overproduction of keratin can cause keratosis pilaris, often blocking the opening of hair follicles and resulting in further skin irritation. These results may therefore also demonstrate another nonirritating benefit of sunblock based on PLA-HPGALD NPs.

The ability of the PO/PLA-HPGALD NPs to protect against DNA double-stranded breaks (DSBs) was evaluated in FVB mice.

The dorsal hair of FVB mice was shaved with electric clippers and treated with depilatory cream. One week later, the mice received either PO/PLA-HPGALD NPs, sunscreen, or no treatment followed by dorsal exposure to UV (<NUM> J/m2) one hour after treatment. For cyclobutane pyrimidine dimers (CPDs) staining, dorsal skin flaps were removed five minutes after UV exposure, and incubated in PBS containing <NUM> EDTA for <NUM> hours at <NUM> to allow separation of the epidermis from the dermis. The epidermal sheet was then rinsed in PBS, fixed in acetone for <NUM> at -<NUM>, then permeabilized in cold PBS containing <NUM>% Triton X-<NUM> for <NUM>. Sheets were denatured with <NUM> NaOH in <NUM>% ethanol for <NUM> and then washed with cold PBS containing <NUM>% Triton X-<NUM> four times, eight min each. Sheets were blocked with PBS containing <NUM>% BSA, <NUM>% Triton-X-<NUM> and <NUM>% goat serum for one hour at room temp, then stained overnight at <NUM> with anti-thymine dimer (<NUM>/ml, Abcam#ab <NUM>) and diluted in PBS containing <NUM>% BSA and <NUM>% Triton X-<NUM>.

The remaining steps were carried out at room temperature. Samples were washed in PBS containing <NUM>% Triton-X <NUM> for two hour, stained for two hour with Alexa568-goat-anti-mouse IgG (Invitrogen), washed again, mounted in DAPI (Invitrogen) and examined under a Leica 5P Confocal microscope.

For γH2AX staining, <NUM> hours after UV exposure, dorsal skin flaps were removed and incubated in <NUM> ammonium thiocyanate for <NUM> at <NUM> to allow for separation of the epidermis from the dermis. The epidermal sheet was then rinsed in PBS, fixed in acetone for <NUM> at -<NUM>, then rehydrated in cold PBS. Sheets were blocked and nuclei were permeabilized in PBS containing <NUM>% BSA and <NUM>% Triton-X-<NUM> for one hour at room temp, then stained overnight at <NUM> with anti-γH2AX (<NUM>/ml, clone JBW30, Millipore, Billerica, MA) and diluted in PBS containing <NUM>% BSA and <NUM>% Triton X-<NUM>.

The remaining steps were carried out at room temperature. Samples were washed in PBS containing <NUM>% Triton-X <NUM> for two hour, stained for two hour with Alexa568-goat-anti-mouse IgG (Invitrogen), washed again, mounted in DAPI (Invitrogen) and examined under a Leica 5P Confocal microscope. For CPD staining, <NUM> fields/sheet (<NUM> sheet/mouse) were taken using the stage control to move <NUM> between fields in a set pattern. The fluorescence from CPD staining on nuclei was quantified by image J. For γH2AX staining, all of the areas with γH2AX+ cells on a sheet (<NUM> sheet / mouse) were imaged. The γH2AX+ cells were counted using Imaged particle analyzer software with the threshold set to eliminate the very faint γH2AX staining. The surface concentration of γH2AX+ cells was calculated by dividing the overall number of the γH2AX+ cells on a sheet with the surface area of the sheet.

Both PO/PLA-HPGALD NPs and sunscreen showed no detectable CPDs, but the positive control (unprotected skin) revealed marked widespread CPD formation after UV exposure (<FIG>). Even though UV filter content in PO/PLA-HPGALD NPs was less than <NUM>% of that in sunscreen, it achieved the same level of UV protection.

DSBs induced by UV irradiation are highly carcinogenic. UVB exposure does not directly produce DSBs (<NPL>)); however, it is possible that UV filters present in the epidermis and dermis can produce ROS after photoactivation, react with cellular DNA, and ultimately produce DSBs (<NPL>); <NPL>); <NPL>); <NPL>); <NPL>); <NPL>)). DSBs recruit phosphorylated histone H2A variant H2AX (γH2AX) to the damaged sites (<NPL>)). The group of mice treated with conventional sunscreen showed the highest level of DNA-damage by γH2AX recruitment; in contrast, the level of γH2AX in both the PO/PLA-HPGALD NPs and non-exposed control were comparable to the normal skin control (<FIG>).

UV exposure from sunlight remains a significant health risk, and there continues to be controversy as to the safety and benefits of commercially available sunscreens. In order to address these issues, a sunblock based on PLA-HPGALD NPs was developed. <FIG> are diagrams comparing application of commercial sunscreen (Sunscreen) to PLA-HPGALD NP (BNPs)-based sunscreen (BNPs/UV filters). <FIG> is a diagram of sunscreen formulations applied onto the skin. <FIG> is a diagram of the skin after application: regular sunscreen penetrates into the skin whereas the PLA-HPGALD NP (BNPs) formulation remains on the stratum corneum. <FIG> is a diagram of the skin after sunlight exposure: UV filters produce deleterious ROS, however, PLA-HPGALD NPs (BNPs) do not penetrate into the skin and prevent ROS mediated toxicity by confining these toxic products within the particle.

Claim 1:
Particles comprising
(i) a core comprising a hydrophobic polymer;
(ii) a shell comprising hyperbranched polyglycerol; and
(iii) one or more therapeutic agents, diagnostic agents, or prophylactic agents encapsulated within the particles;
wherein
the hydrophobic polymer is more hydrophobic than the hyperbranched polyglycerol;
the hyperbranched polyglycerol is covalently bound to the hydrophobic polymer; and
polyethylene glycol is covalently attached to the surface of the particles by converting vicinal diol groups on the hyperbranched polyglycerol to aldehyde groups and reacting the aldehyde groups with functional groups to attach the polyethylene glycol.