Multistage Delivery Technologies: Multifunctional, Interdisciplinary Approaches to Nanomedicine
Authors: Matthew T Haynes, Leaf Huang
Journal: Molecular Therapy (2016)

Nanomedicine frequently employs single particles ranging from very simple1Swenson CE Bolcsak LE Batist G Guthrie THJ Tkaczuk KH Boxenbaum H et al.Pharmacokinetics of doxorubicin administered i.v. as Myocet (TLC D-99; liposome-encapsulated doxorubicin citrate) compared with conventional doxorubicin when given in combination with cyclophosphamide in patients with metastatic breast cancer.Anti-Cancer Drugs. 2003; 14: 239-246Crossref PubMed Scopus (93) Google Scholar to highly multifunctional.2Hu Y Haynes MT Wang Y Liu F Huang L A highly efficient synthetic vector: nonhydrodynamic delivery of DNA to hepatocyte nuclei in vivo.ACS Nano. 2013; 7: 5376-5384Crossref PubMed Scopus (92) Google Scholar However, the diversity of barriers to effective transport can limit the global optimization of delivery for such systems. Multistage particles present an effective means to overcome such issues, allowing scientists to better segment the delivery mechanism into discrete steps by combining multiple formulations for sequential utility in vivo. A recent article in Nature Biotechnology highlights such a system,3Xu R Zhang G Mai J Deng X Segura-Ibarra V Wu S et al.An injectable nanoparticle generator enhances delivery of cancer therapeutics.Nat Biotechnol. 2016; 34: 414-418Crossref PubMed Scopus (224) Google Scholar delivering nanoformulated doxorubicin (Dox) within larger disc-shaped, mesoporous silica particles (MSPs). Following intravenous injection, the silica particles can accumulate in tumor endothelia and release the nanoparticles for uptake and drug release within tumor cells. Such systems raise age-old questions in formulation development4Ferrari M Frontiers in cancer nanomedicine: directing mass transport through biological barriers.Trends Biotechnol. 2010; 28: 181-188Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar,5Blanco E Shen H Ferrari M Principles of nanoparticle design for overcoming biological barriers to drug delivery.Nat Biotechnol. 2015; 33: 941-951Crossref PubMed Scopus (3824) Google Scholar; however, recent work in multifunctional macromolecule,6Akinc A Zumbuehl A Goldberg M Leshchiner ES Busini V Hossain N et al.A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.Nat Biotechnol. 2008; 26: 561-569Crossref PubMed Scopus (932) Google Scholar,7Whitehead KA Dorkin JR Vegas AJ Chang PH Veiseh O Matthews J et al.Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity.Nat Commun. 2014; 5: 4277Crossref PubMed Scopus (323) Google Scholar nanoparticle,2Hu Y Haynes MT Wang Y Liu F Huang L A highly efficient synthetic vector: nonhydrodynamic delivery of DNA to hepatocyte nuclei in vivo.ACS Nano. 2013; 7: 5376-5384Crossref PubMed Scopus (92) Google Scholar,8Liu Y Hu Y Huang L ). Influence of polyethylene glycol density and surface lipid on pharmacokinetics and biodistribution of lipid-calcium-phosphate nanoparticles.Biomaterials. 2014; 35: 3027-3034Crossref PubMed Scopus (72) Google Scholar and cell-based9Klyachko NL Haney MJ Zhao Y Manickam DS Mahajan V Suresh P et al.Macrophages offer a paradigm switch for CNS delivery of therapeutic proteins.Nanomedicine (Lond). 2014; 9: 1403-1422Crossref PubMed Scopus (66) Google Scholar,10Bago JR Alfonso-Pecchio A Okolie O Dumitru R Rinkenbaugh A Baldwin A S et al.Therapeutically engineered induced neural stem cells are tumour-homing and inhibit progression of glioblastoma.Nat Commun. 2016; 7: 10593Crossref PubMed Scopus (87) Google Scholar development should be considered instead, in combination with such formulations and concepts11Huang X Zhang F Wang H Niu G Choi KY Swierczewska M et al.Mesenchymal stem cell-based cell engineering with multifunctional mesoporous silica nanoparticles for tumor delivery.Biomaterials. 2013; 34: 1772-1780Crossref PubMed Scopus (136) Google Scholar to dramatically expand the reach of the drug delivery field as a whole. To succeed, efforts in cancer nanomedicine must surmount an array of barriers to delivery related to processes involved in systemic administration. However, particle-based drug formulations often employ a "one size fits all" approach. Broadly, scientists have tended to focus on singular systems that are either simple to formulate12Lien M-Y Liu L-C Wang H-C Yeh M-H Chen C-J Yeh S-P et al.Safety and efficacy of pegylated liposomal doxorubicin-based adjuvant chemotherapy in patients with stage I–III triple-negative breast cancer.Anticancer Res. 2014; 34: 7319-7326PubMed Google Scholar but limited in performance (e.g., Myocet, a simple liposome encapsulating Dox1Swenson CE Bolcsak LE Batist G Guthrie THJ Tkaczuk KH Boxenbaum H et al.Pharmacokinetics of doxorubicin administered i.v. as Myocet (TLC D-99; liposome-encapsulated doxorubicin citrate) compared with conventional doxorubicin when given in combination with cyclophosphamide in patients with metastatic breast cancer.Anti-Cancer Drugs. 2003; 14: 239-246Crossref PubMed Scopus (93) Google Scholar) or extensively modified with multiple components (polymers, ligands, and other conjugates)2Hu Y Haynes MT Wang Y Liu F Huang L A highly efficient synthetic vector: nonhydrodynamic delivery of DNA to hepatocyte nuclei in vivo.ACS Nano. 2013; 7: 5376-5384Crossref PubMed Scopus (92) Google Scholar,13Haynes MT Huang L Hepatic RNA interference: delivery by synthetic vectors.Drug Deliv Transl Res. 2014; 4: 61-73Crossref PubMed Scopus (9) Google Scholar,14Li S-D Huang L Nanoparticles evading the reticuloendothelial system: role of the supported bilayer.Biochim Biophys Acta. 2009; 1788: 2259-2266Crossref PubMed Scopus (367) Google Scholar that potentially dilute the intrinsic impact of each functional moiety. Both approaches can sacrifice a global optimization of various dynamic processes in systemic delivery, including but not limited to drug encapsulation, particle stability, organ distribution, tissue penetration, cell internalization, and molecular release.15Huang L Liu Y In vivo delivery of RNAi with lipid-based nanoparticles.Ann Rev Biomed Eng. 2011; 13: 507-530Crossref PubMed Scopus (140) Google Scholar These considerations reflect the similar yet distinct biological barriers to such delivery as well, including enzymatic degradation, reticuloendothelial clearance, vascular permeation, interstitial impedance, and molecular efflux.4Ferrari M Frontiers in cancer nanomedicine: directing mass transport through biological barriers.Trends Biotechnol. 2010; 28: 181-188Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar Each perspective informs formulation development, and through such analysis, cancer nanomedicine can be viewed uncommonly as a multiscale mass transfer problem.4Ferrari M Frontiers in cancer nanomedicine: directing mass transport through biological barriers.Trends Biotechnol. 2010; 28: 181-188Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar Thus, rather than focusing on what one particle can do to overcome each heterogeneous barrier, selecting at specific barriers the relevant delivery modality better accommodates the diverse physical phenomena occurring at each stage of delivery, with the smooth coalescence of such systems inevitably benefiting overall efficacy. Such formulations, termed multistage particles, begin to address the single-particle inadequacy by subdividing delivery into discrete and sequential formulations, reducing the codependencies (e.g., between initial particle stability and terminal drug release) that single-particle systems confront among diverse barriers. A variety of multistage delivery systems, including MSPs16Tasciotti E Liu X Bhavane R Plant K Leonard AD Price BK et al.Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications.Nat Nanotechnology. 2008; 3: 151-157Crossref PubMed Scopus (572) Google Scholar and biodegradable gelatin nanoparticles,17Wong C Stylianopoulos T Cui J Martin J Chauhan VP Jiang W et al.Multistage nanoparticle delivery system for deep penetration into tumor tissue.Proc Natl Acad Sci USA. 2011; 108: 2426-2431Crossref PubMed Scopus (825) Google Scholar can encapsulate or surface-decorate quantum dots or superparamagnetic iron oxide nanoparticles18Serda RE Mack A Pulikkathara M Zaske AM Chiappini C Fakhoury J et al.Cellular association and assembly of a multi-stage delivery system.Small. 2010; 6: 1329-1340Crossref PubMed Scopus (66) Google Scholar for imaging and deep tumor penetration, and multistage therapy has been explored with satisfying potency in loading PEGylated (polyethylene glycol–coated) paclitaxel micelles19Blanco E Sangai T Hsiao A Ferrati S Bai L Liu X et al.Multistage delivery of chemotherapeutic nanoparticles for breast cancer treatment.Cancer Lett. 2013; 334: 245-252Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar as well as liposomes containing small interfering RNA (siRNA).20Xu R Huang Y Mai J Zhang G Guo X Xia X et al.Multistage vectored siRNA targeting ataxia–telangiectasia mutated for breast cancer therapy.Small. 2013; 9: 1799-1808Crossref PubMed Scopus (55) Google Scholar The new study, published in Nature Biotechnology, introduces a new multistage system (therein referred to as an "injectable nanoparticle generator," or iNPG) based on MSPs used for the therapeutic delivery of Dox as a polyglutamic acid conjugate.3Xu R Zhang G Mai J Deng X Segura-Ibarra V Wu S et al.An injectable nanoparticle generator enhances delivery of cancer therapeutics.Nat Biotechnol. 2016; 34: 414-418Crossref PubMed Scopus (224) Google Scholar Dox is an anthracycline antitumor antibiotic that acts by intercalating into DNA. The authors effectively conjugated Dox monomers to polyglutamic acid (pDox) and encapsulated this stage 2 system into the stage 1 MSPs, loading to an efficient 25–30% at each stage (7–8% Dox loading overall within MSPs). Intravenous delivery allowed passive iNPG accumulation in tumor metastases within the lung through microvascular attachment. Subsequently, pDox nanoparticles were spontaneously formed upon release from MSPs and internalized by tumor cells, releasing Dox in the perinuclear space in a pH-dependent fashion so as to avoid P-glycoprotein-mediated drug efflux pumps in both naive and drug-resistant tumor models (Figure 1). These methods increased drug exposure within the lung to a mass-normalized extent on par with that of liver exposure, while yielding order-of-magnitude decreases in initial transient Dox exposure within the heart, where Dox is known to be particularly toxic. These effects led to an improved therapeutic outcome for the iNPG system as well as a notably increased maximum tolerated dose, as compared with results using Doxil, a PEGylated liposomal formulation of Dox. The favorable results, coupled with a conceptually complex formulation that is nevertheless developmentally straightforward as a whole, have potent implications for clinical development of related technologies. The first multistage particles, introduced nearly a decade ago,16Tasciotti E Liu X Bhavane R Plant K Leonard AD Price BK et al.Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications.Nat Nanotechnology. 2008; 3: 151-157Crossref PubMed Scopus (572) Google Scholar have developed rapidly into advanced systems capable of incorporating an array of both therapeutic19Blanco E Sangai T Hsiao A Ferrati S Bai L Liu X et al.Multistage delivery of chemotherapeutic nanoparticles for breast cancer treatment.Cancer Lett. 2013; 334: 245-252Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar,20Xu R Huang Y Mai J Zhang G Guo X Xia X et al.Multistage vectored siRNA targeting ataxia–telangiectasia mutated for breast cancer therapy.Small. 2013; 9: 1799-1808Crossref PubMed Scopus (55) Google Scholar and diagnostic16Tasciotti E Liu X Bhavane R Plant K Leonard AD Price BK et al.Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications.Nat Nanotechnology. 2008; 3: 151-157Crossref PubMed Scopus (572) Google Scholar,17Wong C Stylianopoulos T Cui J Martin J Chauhan VP Jiang W et al.Multistage nanoparticle delivery system for deep penetration into tumor tissue.Proc Natl Acad Sci USA. 2011; 108: 2426-2431Crossref PubMed Scopus (825) Google Scholar,21Tasciotti E Godin B Martinez JO Chiappini C Bhavane R Liu X et al.Near-infrared imaging method for the in vivo assessment of the biodistribution of nanoporous silicon particles.Mol Imaging. 2011; 10: 56-68PubMed Google Scholar nanotechnologies into the overall carrier design. In particular, MSP delivery systems have been extensively optimized in the oft-neglected areas of particle hemodynamics, margination, and adhesion, in which particle size and shape can strongly influence the frequency of endothelial interactions overall as well as local microvascular adhesion at the tumor.4Ferrari M Frontiers in cancer nanomedicine: directing mass transport through biological barriers.Trends Biotechnol. 2010; 28: 181-188Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar,5Blanco E Shen H Ferrari M Principles of nanoparticle design for overcoming biological barriers to drug delivery.Nat Biotechnol. 2015; 33: 941-951Crossref PubMed Scopus (3824) Google Scholar,22Lee SY Ferrari M Decuzzi P Design of bio-mimetic particles with enhanced vascular interaction.J Biomech. 2009; 42: 1885-1890Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Considerable motivation in this regard stems from complexities and well-known contradictions encountered in multifunctional nanoparticle design, most typically exhibited in the conflict between polymer-mediated steric exclusion for immune evasion (i.e., PEGylation) and specific and active cell internalization (i.e., targeting).4Ferrari M Frontiers in cancer nanomedicine: directing mass transport through biological barriers.Trends Biotechnol. 2010; 28: 181-188Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar,13Haynes MT Huang L Hepatic RNA interference: delivery by synthetic vectors.Drug Deliv Transl Res. 2014; 4: 61-73Crossref PubMed Scopus (9) Google Scholar,15Huang L Liu Y In vivo delivery of RNAi with lipid-based nanoparticles.Ann Rev Biomed Eng. 2011; 13: 507-530Crossref PubMed Scopus (140) Google Scholar Although multifunctional nanoparticle design is frequently disparaged as an illogical compromise in efficacy, such analyses ignore the extraordinary value represented by recent advances in lipid-based delivery systems in particular. The advent of high-throughput nanotechnology, represented most dramatically by the synthetic lipidoids, reveals the strong impact that the physical and chemical properties of lipids can have on the overall efficacy of hepatic transfection.6Akinc A Zumbuehl A Goldberg M Leshchiner ES Busini V Hossain N et al.A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.Nat Biotechnol. 2008; 26: 561-569Crossref PubMed Scopus (932) Google Scholar,23Love KT Mahon KP Levins CG Whitehead KA Querbes W Dorkin JR et al.Lipid-like materials for low-dose, in vivo gene silencing.Proc Natl Acad Sci USA. 2010; 107: 1864-1869Crossref PubMed Scopus (649) Google Scholar Indeed, this dependence of performance on four essential components (length of lipid chains, number of lipid chains per lipid, degree of amine substitution, and pKa of the particle surface), when rationally selected before any experimental evaluation, can lead to a near-perfect predictive capability in mediating in vivo siRNA efficacy.7Whitehead KA Dorkin JR Vegas AJ Chang PH Veiseh O Matthews J et al.Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity.Nat Commun. 2014; 5: 4277Crossref PubMed Scopus (323) Google Scholar Furthermore, our own work on hepatic uptake and gene transfer exhibits anything but a contradiction between PEGylation and active targeting. At sufficiently high degrees of PEGylation (15–20 mol%), bilayer lipid-coated nanoprecipitates show a dramatic shift in tropism away from uptake by resident macrophage populations (i.e., Kupffer cells) to an almost exclusive internalization by primary hepatocytes.8Liu Y Hu Y Huang L ). Influence of polyethylene glycol density and surface lipid on pharmacokinetics and biodistribution of lipid-calcium-phosphate nanoparticles.Biomaterials. 2014; 35: 3027-3034Crossref PubMed Scopus (72) Google Scholar Meanwhile, active targeting via galactose to hepatocyte asialoglycoprotein receptors serves to enhance not only uptake at the cellular level but the overall biodistribution of the nanoparticles as well, facilitating predominant accumulation in the liver (nearly 50% injected dose).8Liu Y Hu Y Huang L ). Influence of polyethylene glycol density and surface lipid on pharmacokinetics and biodistribution of lipid-calcium-phosphate nanoparticles.Biomaterials. 2014; 35: 3027-3034Crossref PubMed Scopus (72) Google Scholar Coupled with promising clinical development in Alnylam Pharmaceuticals's lipid nanoparticle24Coelho T Adams D Silva A Lozeron P Hawkins PN Mant T et al.Safety and efficacy of RNAi therapy for transthyretin amyloidosis.N Engl J Med. 2013; 369: 819-829Crossref PubMed Scopus (721) Google Scholar and GalNAc25Nair JK Willoughby JLS Chan A Charisse K Alam MR Wang Q et al.Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.J Am Chem Soc. 2014; 136: 16958-16961Crossref PubMed Scopus (660) Google Scholar systems, complex, functional, and actively targeted platform development can serve superbly in the proper delivery context to enhance formulation efficacy. Furthermore, the general concept that has long driven the field—that of passive drug accumulation at sites of inflammation (e.g., tumors), and that underpins the above multistage systems—has recently been superseded by advanced, cell-mediated delivery platforms.9Klyachko NL Haney MJ Zhao Y Manickam DS Mahajan V Suresh P et al.Macrophages offer a paradigm switch for CNS delivery of therapeutic proteins.Nanomedicine (Lond). 2014; 9: 1403-1422Crossref PubMed Scopus (66) Google Scholar,10Bago JR Alfonso-Pecchio A Okolie O Dumitru R Rinkenbaugh A Baldwin A S et al.Therapeutically engineered induced neural stem cells are tumour-homing and inhibit progression of glioblastoma.Nat Commun. 2016; 7: 10593Crossref PubMed Scopus (87) Google Scholar,26Roger M Clavreul A Venier-Julienne M-C Passirani C Montero-Menei C Menei P The potential of combinations of drug-loaded nanoparticle systems and adult stem cells for glioma therapy.Biomaterials. 2011; 32: 2106-2116Crossref PubMed Scopus (68) Google Scholar,27Young JS Morshed RA Kim JW Balyasnikova IV Ahmed AU Lesniak MS Advances in stem cells, induced pluripotent stem cells, and engineered cells: delivery vehicles for anti-glioma therapy.Expert Opin Drug Deliv. 2014; 11: 1733-1746Crossref PubMed Scopus (23) Google Scholar Along with an extraordinary ability to overcome the blood–brain barrier,9Klyachko NL Haney MJ Zhao Y Manickam DS Mahajan V Suresh P et al.Macrophages offer a paradigm switch for CNS delivery of therapeutic proteins.Nanomedicine (Lond). 2014; 9: 1403-1422Crossref PubMed Scopus (66) Google Scholar the capabilities of these platforms in "active targeting" to inflamed tissues in vivo more accurately represent the meaning of the phrase than the ligand-based mechanisms that nanoparticles frequently employ. Cell-mediated systems have proved capable of delivering highly complex cargoes such as nanoformulated enzymes (or nanozymes),9Klyachko NL Haney MJ Zhao Y Manickam DS Mahajan V Suresh P et al.Macrophages offer a paradigm switch for CNS delivery of therapeutic proteins.Nanomedicine (Lond). 2014; 9: 1403-1422Crossref PubMed Scopus (66) Google Scholar as well as penetrating rapidly and chemotropically across diverse tissue barriers toward their target site of inflammation.10Bago JR Alfonso-Pecchio A Okolie O Dumitru R Rinkenbaugh A Baldwin A S et al.Therapeutically engineered induced neural stem cells are tumour-homing and inhibit progression of glioblastoma.Nat Commun. 2016; 7: 10593Crossref PubMed Scopus (87) Google Scholar Furthermore, such cell-based systems represent a truly ideal multifunctional delivery system in their ability to self-express a diverse array of therapeutic and diagnostic agents. Although cell-mediated delivery remains in its relative adolescence compared with particle-based technologies, innumerable sufferers of currently intractable diseases await the seminal advances that will drive these systems forward. So, where do these frequently disparate conceptual areas leave the drug delivery field as a whole? In essence, despite complex and extraordinarily cross-disciplinary bases, the theoretical toolbox for formulation scientists has never before been so well stocked, and the coalescence of such technologies is already under way.11Huang X Zhang F Wang H Niu G Choi KY Swierczewska M et al.Mesenchymal stem cell-based cell engineering with multifunctional mesoporous silica nanoparticles for tumor delivery.Biomaterials. 2013; 34: 1772-1780Crossref PubMed Scopus (136) Google Scholar Hierarchical efforts toward tackling one barrier at a time, multiple barriers at once, and the entireties of drug delivery mechanisms in vivo must be developed in concert between multifunctional as well as multistage systems, with more detailed and better-segmented analyses of specific transfer and mass balance at the levels of each discrete barrier.5Blanco E Shen H Ferrari M Principles of nanoparticle design for overcoming biological barriers to drug delivery.Nat Biotechnol. 2015; 33: 941-951Crossref PubMed Scopus (3824) Google Scholar,13Haynes MT Huang L Hepatic RNA interference: delivery by synthetic vectors.Drug Deliv Transl Res. 2014; 4: 61-73Crossref PubMed Scopus (9) Google Scholar,15Huang L Liu Y In vivo delivery of RNAi with lipid-based nanoparticles.Ann Rev Biomed Eng. 2011; 13: 507-530Crossref PubMed Scopus (140) Google Scholar The outlook for the future of nanomedicine remains as strong as ever. Our lab is supported by NIH grants CA149387, CA198999, and DK100664.