NIH Public Access Author Manuscript Chem Eng Prog. Author manuscript; available in PMC 2014 November 03.
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Published in final edited form as: Chem Eng Prog. 2013 March ; 109(3): 25–30.
Engineering biodegradable nanoparticles for drug and gene delivery Junwei Zhang and Mark Saltzman
1. Introduction
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Nanoparticles (NP) are defined as colloidal particles with a size ranging from one to several hundreds of nanometers. Over the past few decades, nanoparticles have transformed the field of drug and gene delivery. NPs are able to carry therapeutic agents, in the form of small molecule drugs, peptides, proteins and genetic materials. NPs can improve the solubility, stability, circulation half-life and biodistribution of the encapsulated agent. Also, NPs with targeting capability can deliver the therapeutic agents to specific tissues/cells and release the cargo in a sustained fashion, thus reducing their systematic toxicity. NPs can also facilitate the passage of therapeutic agents through some biological barriers, such as the blood-brain barrier (BBB). In this review, we will confine our attention to NPs that are composed of biodegradable polymers. Other reviews are available covering nanocarriers of other compositions (for example, liposomes, micelles2, dendrimers3). Figure 1 illustrates the structure of a nanoparticle with multiple functions, including entrapped gene and drug as the main therapeutic agents, enhancing agents to facilitate the main therapeutic agents, cellular targeting agents, organelle specific targeting agents, poly(ethylene glycol)(PEG) coating for enhanced blood circulation, and polymer that is responsive to certain stimuli. In this review, we will discuss the preparation, basic characteristics, matrix chemistry, application and future direction of the biodegradable NPs for drug and gene delivery.
2. Preparation of nanoparticle-based drug/gene delivery system NIH-PA Author Manuscript
Polymeric nanoparticles can be prepared by either preformed polymers or through polymerization of monomers. Hydrophobic polymers are commonly used to prepare NPs. One widely used method is emulsion/solvent evaporation. To load hydrophobic drugs, the polymer and the drugs are dissolved in a water insoluble organic solvent, which is then mixed with water to form an oil in water (o/w) emulsion. NPs are formed after the evaporation of the organic solvent. To load hydrophilic agents, such as DNA, a variation on this approach, using a water/oil/water (w/o/w) double emulsion-solvent evaporation method is often employed.4 Another method of NP preparation is emulsion/solvent diffusion, where partially-water soluble organic solvents are used to dissolve the polymer. After the formation of o/w emulsion, sufficient water is added to the system, leading to the diffusion of the organic solvent into the aqueous phase and the formation of NPs.5 A third method is emulsion/solvent displacement, where water-miscible organic solvents are used to dissolve the polymer, which is then used to
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produce an emulsion upon mixing with an aqueous phase. The quick diffusion of the organic solvent into water leads to the formation of NPs immediately after the mixing.6 Hydrophilic polymers, such as chitosan, can also be used to make NPs. For example, the positively charged chitosan can be mixed with a negatively charged polymer in aqueous phase. The ionic interaction between the two polymers can lead to the formation of NPs.7 NPs can also be prepared by the polymerization of monomers. One common method is emulsion polymerization, where droplets containing monomer and drug are emulsified in the continuous phase to form oil-in-water or water-in-oil emulsion. The polymerization of the monomer in the droplets leads to the formation of NPs.8
3. Basic characteristics of nanoparticles The physicochemical properties of the NP drug delivery system, such as size, shape, surface properties, drug loading and release, are critical determinants of the fate of the delivery vehicles and the efficacy of the treatment. These factors need to be carefully examined in the preparation of NPs.
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Size of the NPs can influence their biodistribution, protein adsorption and mechanism of cell internalization. For example, for spherical particles, those ranging in size from 100–200nm have the longest circulation half-life because they are large enough to avoid the clearance in the liver, but small enough to escape the filtration in the spleen.9 Shape has recently been identified as an equally important factor determining the biodistribution and cell internalization of NPs. The geometry of interaction between a cell and a particle can also determine the rate of internalization. For example, it has been reported that NPs with an aspect ratio of 3 were internalized 4 times faster than NPs with an aspect ratio of 1 by Hela cells, a cervical cancer cell line.10
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Surface properties have a significant influence on the pharmacokinetic, biodistribution and biocompatibility properties of the NPs. For example, unmodified NPs with a hydrophobic surface are quickly cleared from the bloodstream. Once in the blood, proteins bind to these NPs, allowing them to be recognized and eliminated by the macrophages of the mononuclear phagocytic system (MPS). However, after coating with the hydrophilic polymer, poly(ethylene glycol) (PEG), flexible PEG molecules form a hydrophilic layer on the NPs, which can sterically inhibit the adsorption of blood proteins and delay the clearance of the NPs. As a result, PEG coatings can increase the blood circulation time of NPs by several orders of magnitude.11 In addition to PEG, different types of targeting ligands can also be attached on the surface, which will be discussed later. Drug loading is another important factor in the efficacy of the drug/gene delivery system. Higher drug loading is desired as it can reduce the amount of drug carrier used, thus lowering the possible toxicity from the carrier. Drug/genes can often be loaded in NPs through non-covalent interactions, where the agents are either encapsulated in the NPs during the preparation process, or adsorbed on the surface of the NPs after their formation. Alternatively, the therapeutic agents can be covalently bonded to the NPs. In some situations, covalent coupling can increase the loading of water-soluble drugs.12 Other factors
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such as size of the NP, surface modification and matrix material, can also influence drug loading efficiency. Despite the progress made, obtaining sufficient drug loading is still one of the major hurdles for the development of drug/gene delivery systems. Controlled drug release is also important. The most “desirable” pattern of drug release depends on the application. In situations where long-term release of drug is preferred, a rapid initial release (“burst release”) is generally undesirable, so significant effort has been devoted to making NPs that slowly release agents. Drug cargos can be released in several ways, such as desorption of surface adsorbed/linked drug, diffusion through NP matrix, or matrix erosion: it is essential to understand the mechanism of release to produce NPs with the ideal release properties. The method of drug loading has a profound influence on release mechanism and, therefore, release kinetics. For example, drugs encapsulated inside the NPs are usually released slower than those adsorbed on surface and release of entrapped drug kinetics can be tuned by modifying the physicochemical properties of the matrix polymer, such as molecular weight, hydrophobicity and stability. Preparing NPs from stimuliresponsive materials is another important technique to control drug release property, which is discussed later in more detail.
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4. Materials used in engineering biodegradable NPs 4.1. Materials for drug delivery Biodegradable polymers can be either synthetic or natural. Synthetic polymers are widely used for drug delivery due to their versatility, ease of modification, and low batch-to-batch variability. Poly(alkyl cyanoacrylate) (PACA) is one of the earliest biodegradable polymers formed into NPs for drug delivery. It is prepared by the anionic polymerization of alkyl cyanoacrylic monomers. By changing the length of the alkyl chain, the degradation rate of the polymer can be varied.13 PACA NPs have been used in the delivery of anticancer drugs, antibacterial drugs, anti-inflammatory drugs. However, clinical translation of PACA NPs is difficult, especially for PACA with shorter alkyl chains, which degrade into toxic cyanoacetic acid and formaldehyde.
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A group of aliphatic polyesters, poly (lactic acid) (PLA), poly (glycolic acid)(PGA), and especially poly(lactic-co-glycolic acid)(PLGA), are the most widely studied materials for drug delivery. These materials have been approved for clinical use by the US FDA (Food and Drug Administration). These polymers are synthesized through ring-opening polymerization of glycolide and/or lactide or through the biosynthesis using recombinant bacteria.14 Many controlled release products of PLGA are available in the market, such as Lupron Depot® for the treatment of prostate cancer, Somatuline® LA for the treatment of acromegaly and Risperidal® Consta™ for the treatment of psychiatric disorders. One of the advantages of these polymers is their versatility, as they have now been used for the delivery of small molecular drugs, genes, proteins, and many others. However, one limitation for these materials arises from their acidic degradation products, which have the potential to degrade or denature some biological agents, such as protein and DNA, or change the pH in tissues when delivered locally. One way to solve this problem is to co-encapsulate
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excipients, such as magnesium hydroxide, into the NPs to buffer the acidic microenvironment.15
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Poly(caprolactone) (PCL) is another aliphatic polyester, which is prepared from the ringopening polymerization of caprolactone. PCL has a slow degradation rate so it is commonly used in the long-term controlled release systems. Several anti-cancer drugs, insulin and antifungal drugs have been delivery by PCL. Polyanhydrides are a group of biodegradable polymers that can be synthesized through condensation of diacids/diacid esters, ring-opening polymerization of anhydrides, and other methods. The degradation rate of polyanhydrides can be altered from several days to several years by using copolymers of aliphatic and aromatic anhydride monomers at different ratios. One such example is the copolymer of 1,3-bis-(p-carboxyphenoxy) propane and sebacic acid poly(CPP-SA), which was used to fabricate a carmustine-releasing implant (called GLIADEL®), a US FDA approved controlled released device for the treatment for recurrent malignant glioma.16
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Apart from the synthetic polymers, a series of natural polymers and their derivatives have been used in preparing biodegradable NPs. Chitosan is a biodegradable semi-natural polymer prepared by partial N-deacetylation of chitin. Chitosan NPs have been used in the delivery of agents such as anti-hormonal drugs and insulin. The mucosal-adhesive nature of chitosan has suggested its use in mucosal immunization through oral administration.17 Another widely used natural polymer is gelatin, which is obtained by the thermal denaturation of collagen. Gelatin has been used to make NPs for the delivery of proteins, genetic materials or small molecule drugs. Both chitosan and gelatin have excellent biocompatibility. However, they usually suffer due to their high batch-to-batch variability, low synthetic flexibility and risk of viral infection. 4.2. Materials for gene delivery
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Gene therapy—the treatment or prevention of disease through transfer of genetic material— is a promising approach for genetic disorders, neurodegenerative diseases, and cancer. Since unprotected DNA will be degraded by endogenous nucleases, the success of gene therapy is largely dependent on the development of vectors that can selectively and efficiently deliver a gene to target cells with minimal toxicity. Biodegradable polymers have great potential as vectors because of their biocompatibility, flexibility in synthesis, and potential for modification. Polymeric NPs for gene delivery can be formed by simple electrostatic complexation of DNA with polymers, encapsulating DNA into polymeric NPs, or by complexing DNA to the surface of preformed polymeric NPs. PLGA has been used to encapsulate genes and release them in a sustained fashion.18 PLGA NPs are also capable for endolysosomal escape by destabilizing the endosome membrane, which is crucial for gene transfection because genes trapped in the endolysosomes will be degraded by the low pH in these vesicles. Polyalkylcyanacrylate (PACA) has also been used for gene delivery, where genes are complexed at the surface of preformed NPs.19
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Cationic polymers are widely used for gene delivery due to their ability to electrostatically complex with DNA. However, many cationic polymers, such as polyethylenimine(PEI) and poly(l-lysine)(PLL), are non-biodegradable and have high toxicity.20, 21 To solve this problem, a group of biodegradable polycations, poly(β-amino ester)s or PBAEs, have been synthesized for gene delivery. They are prepared by the addition of either primary or bissecondary aliphatic amine to diacrylate esters. These polymers can electrostatically interact with DNA (and other polynucleotides) through the tertiary amines in the backbone and they exhibit less toxicity than PEI. 22 A novel group of poly (amine-co-ester)s were recently synthesized via lipase catalyzed polymerization of lactone, diester and amino diols. These polymers have low cationic charge, thus they show minimal toxicity; also, they have very high molecular weight and high hydrophobicity, which can facilitate their complexation with DNA. These polymers are among the most efficient polymeric gene vectors yet reported.23 Natural polymers such as chitosan and gelatin have also been used for gene delivery. 4.3. Research on new materials for drug and gene delivery
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Although various polymers have been engineered into NPs for drug/gene delivery, in many cases they failed to significantly improve the effectiveness of the drugs/genes for clinical application. Novel materials are still needed to address some of the major barriers in this field, such as low drug loading efficiency into the NP and the uncontrolled drug release. Multi-functionality is crucial for addressing the multiple barriers for drug and gene delivery. For example, an efficient gene vector should be able to overcome the various extracellular and intracellular barriers, such as gene packaging, stability in serum, cell-specific targeting, endolysosomal escape, nuclear localization, and gene unpackaging.24 As an attempt to address these barriers, an octa-functional PLGA NP for siRNA delivery was recently described25, in which functional components for endosomal escape, enhancement of siRNA potency, increase serum-stability, cell-penetration and tumor targeting were incorporated.
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Another interesting area is stimuli-responsive polymer, which can be degraded or destabilized in the presence of certain stimuli, which activates the release of loaded cargo. Different types of stimuli, both internal and external, have been employed in the design of NPs for drug/gene delivery. One common type of internal stimuli is pH change. For example, endosomal/lysosomal environments and many solid tumors are acidic, when compared to the normal tissue pH of 7.4. By introducing pH-sensitive bonds in the polymer matrix or in the linker between the matrix and the cargo, NPs loaded cargos can be released in response to acidic microenvironments. 26 Redox potential is another type of internal stimulus. Redox sensitive groups, such as disulfide, can be incorporated into the NP-based delivery system. Since the cytosol has a higher reducing potential than the intercellular matrix, the disulfide bonds will be degraded and the cargo will be released into the cytosol.27 Enzyme-sensitivity is also employed in the stimuli-responsive polymers. For example, peptides sensitive to matrix metalloproteinases (MMPs), an enzyme overexpressed in tumors, have been used to link drugs to NPs 28. Once these NP accumulate in tumors, the
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linker is degraded and the loaded drug released. Materials that are responsive to external stimuli, such as magnetic field, ultrasound, heat, have also been engineered to form NPs.
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Although significant advances have been made in design of stimuli-responsive polymers, most of these polymers are non-biodegradable; efforts are still needed to produce biodegradable polymer for this application.
5. Important applications of engineering biodegradable nanoparticles in gene/drug delivery 5.1. Improving the pharmacokinetic properties of therapeutic agents
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NPs can significantly improve the pharmacokinetic properties of encapsulated agents. First, by encapsulating into NPs, fragile biological agents, such as DNA, can be protected against premature degradation. Second, hydrophobic drugs, such as the anticancer drug camptothecin, can be encapsulated into NPs and their stability in blood circulation can be greatly improved.29 Also, by coating the NP with PEG, the circulation time of the drugloaded NPs will be greatly improved30, which is crucial for sustained release and controlled biodistribution. A recent example of this approach is the encapsulation of docetaxel in PLGA-PEG NPs for treatment of solid tumors.31 5.2. Controlled release of therapeutic agents One goal in designing NPs for drug/gene delivery is to lower toxicity. This can be achieved by controlling the drug release pattern from the NP, both temporally and spatially. In terms of temporal control, the physicochemical properties of the polymer are adjusted to achieve a sustained release rate that maintains drug levels within the therapeutic window for prolonged periods. The release rate can also be tuned to match the life cycle of specific cells to reach an improved efficacy.
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Spatial control is best illustrated by targeting cancer cells (Figure 2). The targeting techniques include passive targeting and active targeting. Since tumor vasculature is underdeveloped and leaky, NPs can leak out and accumulate in the tumor interstitial space. The poor lymphatic drainage in tumors further enhances the accumulation of NPs. This phenomenon is called the enhanced permeability and retention effect (EPR) and is the basis for passive tumor targeting. Active tumor targeting employs specific ligands to target the surface receptors over-expressed on tumor cells. Transferrin and folic acid are widely used targeting ligands because many types of cancer cells over-express transferrin or folic acid receptors. Antibodies have also been used as highly selective targeting ligands, such as the antibody targeting prostate-specific membrane antigen (PSMA). Short peptides capable of binding to cell surface receptors, such as the arginine-glycine-asparitic acid (RGD) peptide expressed on angiogenic endothelial cells, are also used. Recent advances have been made in the area of organelle-specific targeting. The localization of the cargo in specific organelles can be crucial for some agents. For example, in the case of gene delivery, many genetic materials can only be effective if they localize in nucleus. Recent advances have been made by conjugating a short peptide sequences, called nuclear
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localization signals (NLS), to the NPs 32. Targeted delivery to mitochondria has also been studied.
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The above mentioned stimuli-responsive materials can also be combined with the passive and active targeting methods to allow a more accurate control over drug release pattern. 5.3. Cross biological barriers Another potential role of NPs is to deliver agents through biological barriers. One example is transport through the blood-brain barrier (BBB). The transport of substances between the circulation and the central nervous system is restricted by the BBB, which makes it the major barrier for the delivery of agents into the brain though systematic administration. NPs might facilitate transport through the BBB by binding to endothelial cells via receptors that lead to transport of the NP through the cell and into the brain. 33 While some intriguing examples of this approach have been reported, it is not yet clear that sufficient numbers of NPs are transported to make the approach generally useful.
6. Future directions NIH-PA Author Manuscript
Multi-functional NPs offer the potential for high drug loading, activated release, and flexibile design to address the need of different diseases. NPs that are able to carry and release multiple drugs, deploy specific targeting ligands, and retain imaging contrast agents will be useful in addressing some of the most pressing clinical problems, such as the treatment of metastatic cancer. NPs are also needed for efficient gene delivery as the lack of proper vectors seriously hinders the clinical application of gene therapy. Biomimetism is a promising direction as nature has already designed the most efficient gene vector-virus. The use of combinational chemistry for the discovery of novel materials presents another promising method. Knowledge in chemistry and biology is vital for the future development of drug/gene delivery NPs. It is essential to understand the physiochemical structure that determine the properties of NPs, as well as the molecular interactions that govern how NPs interact with cells and tissues. Further, the unique characteristics of each human disease must be appreciated, in order to design NPs that behave with optimal effectiveness in that setting.
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References 1. Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nature Reviews Drug Discovery. 2005; 4(2):145–160. 2. Liggins RT, Burt HM. Polyether-polyester diblock copolymers for the preparation of paclitaxel loaded polymeric micelle formulations. Adv Drug Deliv Rev. 2002; 54(2):191–202. [PubMed: 11897145] 3. Lee CC, et al. Designing dendrimers for biological applications. Nat Biotechnol. 2005; 23(12): 1517–1526. [PubMed: 16333296] 4. Zambaux MF, et al. Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method. Journal of Controlled Release. 1998; 50(1–3): 31–40. [PubMed: 9685870] 5. Vargas A, et al. Improved photodynamic activity of porphyrin loaded into nanoparticles: an in vivo evaluation using chick embryos. Int J Pharm. 2004; 286(1–2):131–145. [PubMed: 15501010] Chem Eng Prog. Author manuscript; available in PMC 2014 November 03.
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6. Barichello JM, et al. Encapsulation of hydrophilic and lipophilic drugs in PLGA nanoparticles by the nanoprecipitation method. Drug Development and Industrial Pharmacy. 1999; 25(4):471–476. [PubMed: 10194602] 7. Calvo P, et al. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. Journal of Applied Polymer Science. 1997; 63(1):125–132. 8. Ekman B, Sjoholm I. Improved Stability of Proteins Immobilized in Microparticles Prepared by a Modified Emulsion Polymerization Technique. J Pharm Sci. 1978; 67(5):693–696. [PubMed: 417171] 9. Petros RA, DeSimone JM. Strategies in the design of nanoparticles for therapeutic applications. Nature Reviews Drug Discovery. 2010; 9(8):615–627. 10. Gratton SEA, et al. The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci U S A. 2008; 105(33):11613–11618. [PubMed: 18697944] 11. Gref R, et al. Biodegradable Long-Circulating Polymeric Nanospheres. Science. 1994; 263(5153): 1600–1603. [PubMed: 8128245] 12. Yoo HS, et al. Biodegradable nanoparticles containing doxorubicin-PLGA conjugate for sustained release. Pharm Res. 1999; 16(7):1114–1118. [PubMed: 10450940] 13. Couvreur P, et al. Adsorption of antineoplastic drugs to polyalkylcyanoacrylate nanoparticles and their release in calf serum. J Pharm Sci. 1979; 68(12):1521–1524. [PubMed: 529043] 14. Yang TH, et al. Biosynthesis of Polylactic Acid and Its Copolymers Using Evolved Propionate CoA Transferase and PHA Synthase. Biotechnol Bioeng. 2010; 105(1):150–160. [PubMed: 19937726] 15. Zhu G, et al. Stabilization of proteins encapsulated in injectable poly (lactide- co-glycolide). Nat Biotechnol. 2000; 18(1):52–57. [PubMed: 10625391] 16. Fleming AB, Saltzman WM. Pharmacokinetics of the carmustine implant. Clin Pharmacokinet. 2002; 41(6):403–419. [PubMed: 12074689] 17. Ferrari F, et al. Characterization of rheological and mucoadhesive properties of three grades of chitosan hydrochloride. Farmaco. 1997; 52(6–7):493–497. [PubMed: 9372602] 18. Panyam J, et al. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. Faseb Journal. 2002; 16(10) 19. Couvreur P, et al. Polycyanoacrylate nanocapsules as potential lysosomotropic carriers: preparation, morphological and sorptive properties. J Pharm Pharmacol. 1979; 31(5):331–332. [PubMed: 37304] 20. Jeong JH, et al. DNA transfection using linear poly(ethylenimine) prepared by controlled acid hydrolysis of poly(2-ethyl-2-oxazoline). J Control Release. 2001; 73(2–3):391–399. [PubMed: 11516514] 21. Choi YH, et al. Polyethylene glycol-grafted poly-L-lysine as polymeric gene carrier. Journal of Controlled Release. 1998; 54(1):39–48. [PubMed: 9741902] 22. Lynn DM, Langer R. Degradable poly(beta-amino esters): Synthesis, characterization, and selfassembly with plasmid DNA. J Am Chem Soc. 2000; 122(44):10761–10768. 23. Zhou J, et al. Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery. Nat Mater. 2012; 11(1):82–90. [PubMed: 22138789] 24. Pack DW, et al. Design and development of polymers for gene delivery. Nat Rev Drug Discov. 2005; 4(7):581–593. [PubMed: 16052241] 25. Zhou J, et al. Octa-functional PLGA nanoparticles for targeted and efficient siRNA delivery to tumors. Biomaterials. 2012; 33(2):583–591. [PubMed: 22014944] 26. Shenoy D, et al. Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pHsensitive system for tumor-targeted delivery of hydrophobic drugs: Part 2. In vivo distribution and tumor localization studies. Pharm Res. 2005; 22(12):2107–2114. [PubMed: 16254763] 27. Cavallaro G, et al. Reversibly stable thiopolyplexes for intracellular delivery of genes. Journal of Controlled Release. 2006; 115(3):322–334. [PubMed: 17028038] 28. Chau Y, et al. Antitumor efficacy of a novel polymer-peptide-drug conjugate in human tumor xenograft models. International Journal of Cancer. 2006; 118(6):1519–1526.
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29. Liu J, et al. Poly(omega-pentadecalactone-co-butylene-co-succinate) nanoparticles as biodegradable carriers for camptothecin delivery. Biomaterials. 2009; 30(29):5707–5719. [PubMed: 19632718] 30. Gref R, et al. Biodegradable long-circulating polymeric nanospheres. Science. 1994; 263(5153): 1600–1603. [PubMed: 8128245] 31. Hrkach J, et al. Preclinical development and clinical translation of a PSMA-targeted docetaxel nanoparticle with a differentiated pharmacological profile. Sci Transl Med. 2012; 4(128): 128ra139. 32. Misra R, Sahoo SK. Intracellular trafficking of nuclear localization signal conjugated nanoparticles for cancer therapy. European Journal of Pharmaceutical Sciences. 2010; 39(1–3):152–163. [PubMed: 19961929] 33. Patel T, et al. Polymeric nanoparticles for drug delivery to the central nervous system. Adv Drug Deliv Rev. 2012; 64(7):701–705. [PubMed: 22210134]
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Figure 1.
Multi-functional nanoparticles for drug/gene delivery
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NIH-PA Author Manuscript Figure 2.
Tumor targeting NPs. Showing passive (EPR effect) and active targeting with tumor targeting ligands.
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Published in final edited form as: Chem Eng Prog. 2013 March ; 109(3): 25–30.
Engineering biodegradable nanoparticles for drug and gene delivery Junwei Zhang and Mark Saltzman
1. Introduction
NIH-PA Author Manuscript
Nanoparticles (NP) are defined as colloidal particles with a size ranging from one to several hundreds of nanometers. Over the past few decades, nanoparticles have transformed the field of drug and gene delivery. NPs are able to carry therapeutic agents, in the form of small molecule drugs, peptides, proteins and genetic materials. NPs can improve the solubility, stability, circulation half-life and biodistribution of the encapsulated agent. Also, NPs with targeting capability can deliver the therapeutic agents to specific tissues/cells and release the cargo in a sustained fashion, thus reducing their systematic toxicity. NPs can also facilitate the passage of therapeutic agents through some biological barriers, such as the blood-brain barrier (BBB). In this review, we will confine our attention to NPs that are composed of biodegradable polymers. Other reviews are available covering nanocarriers of other compositions (for example, liposomes, micelles2, dendrimers3). Figure 1 illustrates the structure of a nanoparticle with multiple functions, including entrapped gene and drug as the main therapeutic agents, enhancing agents to facilitate the main therapeutic agents, cellular targeting agents, organelle specific targeting agents, poly(ethylene glycol)(PEG) coating for enhanced blood circulation, and polymer that is responsive to certain stimuli. In this review, we will discuss the preparation, basic characteristics, matrix chemistry, application and future direction of the biodegradable NPs for drug and gene delivery.
2. Preparation of nanoparticle-based drug/gene delivery system NIH-PA Author Manuscript
Polymeric nanoparticles can be prepared by either preformed polymers or through polymerization of monomers. Hydrophobic polymers are commonly used to prepare NPs. One widely used method is emulsion/solvent evaporation. To load hydrophobic drugs, the polymer and the drugs are dissolved in a water insoluble organic solvent, which is then mixed with water to form an oil in water (o/w) emulsion. NPs are formed after the evaporation of the organic solvent. To load hydrophilic agents, such as DNA, a variation on this approach, using a water/oil/water (w/o/w) double emulsion-solvent evaporation method is often employed.4 Another method of NP preparation is emulsion/solvent diffusion, where partially-water soluble organic solvents are used to dissolve the polymer. After the formation of o/w emulsion, sufficient water is added to the system, leading to the diffusion of the organic solvent into the aqueous phase and the formation of NPs.5 A third method is emulsion/solvent displacement, where water-miscible organic solvents are used to dissolve the polymer, which is then used to
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produce an emulsion upon mixing with an aqueous phase. The quick diffusion of the organic solvent into water leads to the formation of NPs immediately after the mixing.6 Hydrophilic polymers, such as chitosan, can also be used to make NPs. For example, the positively charged chitosan can be mixed with a negatively charged polymer in aqueous phase. The ionic interaction between the two polymers can lead to the formation of NPs.7 NPs can also be prepared by the polymerization of monomers. One common method is emulsion polymerization, where droplets containing monomer and drug are emulsified in the continuous phase to form oil-in-water or water-in-oil emulsion. The polymerization of the monomer in the droplets leads to the formation of NPs.8
3. Basic characteristics of nanoparticles The physicochemical properties of the NP drug delivery system, such as size, shape, surface properties, drug loading and release, are critical determinants of the fate of the delivery vehicles and the efficacy of the treatment. These factors need to be carefully examined in the preparation of NPs.
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Size of the NPs can influence their biodistribution, protein adsorption and mechanism of cell internalization. For example, for spherical particles, those ranging in size from 100–200nm have the longest circulation half-life because they are large enough to avoid the clearance in the liver, but small enough to escape the filtration in the spleen.9 Shape has recently been identified as an equally important factor determining the biodistribution and cell internalization of NPs. The geometry of interaction between a cell and a particle can also determine the rate of internalization. For example, it has been reported that NPs with an aspect ratio of 3 were internalized 4 times faster than NPs with an aspect ratio of 1 by Hela cells, a cervical cancer cell line.10
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Surface properties have a significant influence on the pharmacokinetic, biodistribution and biocompatibility properties of the NPs. For example, unmodified NPs with a hydrophobic surface are quickly cleared from the bloodstream. Once in the blood, proteins bind to these NPs, allowing them to be recognized and eliminated by the macrophages of the mononuclear phagocytic system (MPS). However, after coating with the hydrophilic polymer, poly(ethylene glycol) (PEG), flexible PEG molecules form a hydrophilic layer on the NPs, which can sterically inhibit the adsorption of blood proteins and delay the clearance of the NPs. As a result, PEG coatings can increase the blood circulation time of NPs by several orders of magnitude.11 In addition to PEG, different types of targeting ligands can also be attached on the surface, which will be discussed later. Drug loading is another important factor in the efficacy of the drug/gene delivery system. Higher drug loading is desired as it can reduce the amount of drug carrier used, thus lowering the possible toxicity from the carrier. Drug/genes can often be loaded in NPs through non-covalent interactions, where the agents are either encapsulated in the NPs during the preparation process, or adsorbed on the surface of the NPs after their formation. Alternatively, the therapeutic agents can be covalently bonded to the NPs. In some situations, covalent coupling can increase the loading of water-soluble drugs.12 Other factors
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such as size of the NP, surface modification and matrix material, can also influence drug loading efficiency. Despite the progress made, obtaining sufficient drug loading is still one of the major hurdles for the development of drug/gene delivery systems. Controlled drug release is also important. The most “desirable” pattern of drug release depends on the application. In situations where long-term release of drug is preferred, a rapid initial release (“burst release”) is generally undesirable, so significant effort has been devoted to making NPs that slowly release agents. Drug cargos can be released in several ways, such as desorption of surface adsorbed/linked drug, diffusion through NP matrix, or matrix erosion: it is essential to understand the mechanism of release to produce NPs with the ideal release properties. The method of drug loading has a profound influence on release mechanism and, therefore, release kinetics. For example, drugs encapsulated inside the NPs are usually released slower than those adsorbed on surface and release of entrapped drug kinetics can be tuned by modifying the physicochemical properties of the matrix polymer, such as molecular weight, hydrophobicity and stability. Preparing NPs from stimuliresponsive materials is another important technique to control drug release property, which is discussed later in more detail.
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4. Materials used in engineering biodegradable NPs 4.1. Materials for drug delivery Biodegradable polymers can be either synthetic or natural. Synthetic polymers are widely used for drug delivery due to their versatility, ease of modification, and low batch-to-batch variability. Poly(alkyl cyanoacrylate) (PACA) is one of the earliest biodegradable polymers formed into NPs for drug delivery. It is prepared by the anionic polymerization of alkyl cyanoacrylic monomers. By changing the length of the alkyl chain, the degradation rate of the polymer can be varied.13 PACA NPs have been used in the delivery of anticancer drugs, antibacterial drugs, anti-inflammatory drugs. However, clinical translation of PACA NPs is difficult, especially for PACA with shorter alkyl chains, which degrade into toxic cyanoacetic acid and formaldehyde.
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A group of aliphatic polyesters, poly (lactic acid) (PLA), poly (glycolic acid)(PGA), and especially poly(lactic-co-glycolic acid)(PLGA), are the most widely studied materials for drug delivery. These materials have been approved for clinical use by the US FDA (Food and Drug Administration). These polymers are synthesized through ring-opening polymerization of glycolide and/or lactide or through the biosynthesis using recombinant bacteria.14 Many controlled release products of PLGA are available in the market, such as Lupron Depot® for the treatment of prostate cancer, Somatuline® LA for the treatment of acromegaly and Risperidal® Consta™ for the treatment of psychiatric disorders. One of the advantages of these polymers is their versatility, as they have now been used for the delivery of small molecular drugs, genes, proteins, and many others. However, one limitation for these materials arises from their acidic degradation products, which have the potential to degrade or denature some biological agents, such as protein and DNA, or change the pH in tissues when delivered locally. One way to solve this problem is to co-encapsulate
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excipients, such as magnesium hydroxide, into the NPs to buffer the acidic microenvironment.15
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Poly(caprolactone) (PCL) is another aliphatic polyester, which is prepared from the ringopening polymerization of caprolactone. PCL has a slow degradation rate so it is commonly used in the long-term controlled release systems. Several anti-cancer drugs, insulin and antifungal drugs have been delivery by PCL. Polyanhydrides are a group of biodegradable polymers that can be synthesized through condensation of diacids/diacid esters, ring-opening polymerization of anhydrides, and other methods. The degradation rate of polyanhydrides can be altered from several days to several years by using copolymers of aliphatic and aromatic anhydride monomers at different ratios. One such example is the copolymer of 1,3-bis-(p-carboxyphenoxy) propane and sebacic acid poly(CPP-SA), which was used to fabricate a carmustine-releasing implant (called GLIADEL®), a US FDA approved controlled released device for the treatment for recurrent malignant glioma.16
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Apart from the synthetic polymers, a series of natural polymers and their derivatives have been used in preparing biodegradable NPs. Chitosan is a biodegradable semi-natural polymer prepared by partial N-deacetylation of chitin. Chitosan NPs have been used in the delivery of agents such as anti-hormonal drugs and insulin. The mucosal-adhesive nature of chitosan has suggested its use in mucosal immunization through oral administration.17 Another widely used natural polymer is gelatin, which is obtained by the thermal denaturation of collagen. Gelatin has been used to make NPs for the delivery of proteins, genetic materials or small molecule drugs. Both chitosan and gelatin have excellent biocompatibility. However, they usually suffer due to their high batch-to-batch variability, low synthetic flexibility and risk of viral infection. 4.2. Materials for gene delivery
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Gene therapy—the treatment or prevention of disease through transfer of genetic material— is a promising approach for genetic disorders, neurodegenerative diseases, and cancer. Since unprotected DNA will be degraded by endogenous nucleases, the success of gene therapy is largely dependent on the development of vectors that can selectively and efficiently deliver a gene to target cells with minimal toxicity. Biodegradable polymers have great potential as vectors because of their biocompatibility, flexibility in synthesis, and potential for modification. Polymeric NPs for gene delivery can be formed by simple electrostatic complexation of DNA with polymers, encapsulating DNA into polymeric NPs, or by complexing DNA to the surface of preformed polymeric NPs. PLGA has been used to encapsulate genes and release them in a sustained fashion.18 PLGA NPs are also capable for endolysosomal escape by destabilizing the endosome membrane, which is crucial for gene transfection because genes trapped in the endolysosomes will be degraded by the low pH in these vesicles. Polyalkylcyanacrylate (PACA) has also been used for gene delivery, where genes are complexed at the surface of preformed NPs.19
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Cationic polymers are widely used for gene delivery due to their ability to electrostatically complex with DNA. However, many cationic polymers, such as polyethylenimine(PEI) and poly(l-lysine)(PLL), are non-biodegradable and have high toxicity.20, 21 To solve this problem, a group of biodegradable polycations, poly(β-amino ester)s or PBAEs, have been synthesized for gene delivery. They are prepared by the addition of either primary or bissecondary aliphatic amine to diacrylate esters. These polymers can electrostatically interact with DNA (and other polynucleotides) through the tertiary amines in the backbone and they exhibit less toxicity than PEI. 22 A novel group of poly (amine-co-ester)s were recently synthesized via lipase catalyzed polymerization of lactone, diester and amino diols. These polymers have low cationic charge, thus they show minimal toxicity; also, they have very high molecular weight and high hydrophobicity, which can facilitate their complexation with DNA. These polymers are among the most efficient polymeric gene vectors yet reported.23 Natural polymers such as chitosan and gelatin have also been used for gene delivery. 4.3. Research on new materials for drug and gene delivery
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Although various polymers have been engineered into NPs for drug/gene delivery, in many cases they failed to significantly improve the effectiveness of the drugs/genes for clinical application. Novel materials are still needed to address some of the major barriers in this field, such as low drug loading efficiency into the NP and the uncontrolled drug release. Multi-functionality is crucial for addressing the multiple barriers for drug and gene delivery. For example, an efficient gene vector should be able to overcome the various extracellular and intracellular barriers, such as gene packaging, stability in serum, cell-specific targeting, endolysosomal escape, nuclear localization, and gene unpackaging.24 As an attempt to address these barriers, an octa-functional PLGA NP for siRNA delivery was recently described25, in which functional components for endosomal escape, enhancement of siRNA potency, increase serum-stability, cell-penetration and tumor targeting were incorporated.
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Another interesting area is stimuli-responsive polymer, which can be degraded or destabilized in the presence of certain stimuli, which activates the release of loaded cargo. Different types of stimuli, both internal and external, have been employed in the design of NPs for drug/gene delivery. One common type of internal stimuli is pH change. For example, endosomal/lysosomal environments and many solid tumors are acidic, when compared to the normal tissue pH of 7.4. By introducing pH-sensitive bonds in the polymer matrix or in the linker between the matrix and the cargo, NPs loaded cargos can be released in response to acidic microenvironments. 26 Redox potential is another type of internal stimulus. Redox sensitive groups, such as disulfide, can be incorporated into the NP-based delivery system. Since the cytosol has a higher reducing potential than the intercellular matrix, the disulfide bonds will be degraded and the cargo will be released into the cytosol.27 Enzyme-sensitivity is also employed in the stimuli-responsive polymers. For example, peptides sensitive to matrix metalloproteinases (MMPs), an enzyme overexpressed in tumors, have been used to link drugs to NPs 28. Once these NP accumulate in tumors, the
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linker is degraded and the loaded drug released. Materials that are responsive to external stimuli, such as magnetic field, ultrasound, heat, have also been engineered to form NPs.
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Although significant advances have been made in design of stimuli-responsive polymers, most of these polymers are non-biodegradable; efforts are still needed to produce biodegradable polymer for this application.
5. Important applications of engineering biodegradable nanoparticles in gene/drug delivery 5.1. Improving the pharmacokinetic properties of therapeutic agents
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NPs can significantly improve the pharmacokinetic properties of encapsulated agents. First, by encapsulating into NPs, fragile biological agents, such as DNA, can be protected against premature degradation. Second, hydrophobic drugs, such as the anticancer drug camptothecin, can be encapsulated into NPs and their stability in blood circulation can be greatly improved.29 Also, by coating the NP with PEG, the circulation time of the drugloaded NPs will be greatly improved30, which is crucial for sustained release and controlled biodistribution. A recent example of this approach is the encapsulation of docetaxel in PLGA-PEG NPs for treatment of solid tumors.31 5.2. Controlled release of therapeutic agents One goal in designing NPs for drug/gene delivery is to lower toxicity. This can be achieved by controlling the drug release pattern from the NP, both temporally and spatially. In terms of temporal control, the physicochemical properties of the polymer are adjusted to achieve a sustained release rate that maintains drug levels within the therapeutic window for prolonged periods. The release rate can also be tuned to match the life cycle of specific cells to reach an improved efficacy.
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Spatial control is best illustrated by targeting cancer cells (Figure 2). The targeting techniques include passive targeting and active targeting. Since tumor vasculature is underdeveloped and leaky, NPs can leak out and accumulate in the tumor interstitial space. The poor lymphatic drainage in tumors further enhances the accumulation of NPs. This phenomenon is called the enhanced permeability and retention effect (EPR) and is the basis for passive tumor targeting. Active tumor targeting employs specific ligands to target the surface receptors over-expressed on tumor cells. Transferrin and folic acid are widely used targeting ligands because many types of cancer cells over-express transferrin or folic acid receptors. Antibodies have also been used as highly selective targeting ligands, such as the antibody targeting prostate-specific membrane antigen (PSMA). Short peptides capable of binding to cell surface receptors, such as the arginine-glycine-asparitic acid (RGD) peptide expressed on angiogenic endothelial cells, are also used. Recent advances have been made in the area of organelle-specific targeting. The localization of the cargo in specific organelles can be crucial for some agents. For example, in the case of gene delivery, many genetic materials can only be effective if they localize in nucleus. Recent advances have been made by conjugating a short peptide sequences, called nuclear
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localization signals (NLS), to the NPs 32. Targeted delivery to mitochondria has also been studied.
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The above mentioned stimuli-responsive materials can also be combined with the passive and active targeting methods to allow a more accurate control over drug release pattern. 5.3. Cross biological barriers Another potential role of NPs is to deliver agents through biological barriers. One example is transport through the blood-brain barrier (BBB). The transport of substances between the circulation and the central nervous system is restricted by the BBB, which makes it the major barrier for the delivery of agents into the brain though systematic administration. NPs might facilitate transport through the BBB by binding to endothelial cells via receptors that lead to transport of the NP through the cell and into the brain. 33 While some intriguing examples of this approach have been reported, it is not yet clear that sufficient numbers of NPs are transported to make the approach generally useful.
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Multi-functional NPs offer the potential for high drug loading, activated release, and flexibile design to address the need of different diseases. NPs that are able to carry and release multiple drugs, deploy specific targeting ligands, and retain imaging contrast agents will be useful in addressing some of the most pressing clinical problems, such as the treatment of metastatic cancer. NPs are also needed for efficient gene delivery as the lack of proper vectors seriously hinders the clinical application of gene therapy. Biomimetism is a promising direction as nature has already designed the most efficient gene vector-virus. The use of combinational chemistry for the discovery of novel materials presents another promising method. Knowledge in chemistry and biology is vital for the future development of drug/gene delivery NPs. It is essential to understand the physiochemical structure that determine the properties of NPs, as well as the molecular interactions that govern how NPs interact with cells and tissues. Further, the unique characteristics of each human disease must be appreciated, in order to design NPs that behave with optimal effectiveness in that setting.
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Figure 1.
Multi-functional nanoparticles for drug/gene delivery
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Tumor targeting NPs. Showing passive (EPR effect) and active targeting with tumor targeting ligands.
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