Review pubs.acs.org/CR
Biodegradable Polymer Nanogels for Drug/Nucleic Acid Delivery Yulin Li,*,†,‡ Dina Maciel,† Joaõ Rodrigues,*,† Xiangyang Shi,*,†,§ and Helena Tomás*,† †
CQM-Centro de Química da Madeira, MMRG, Universidade da Madeira, Campus da Penteada 9000-390, Funchal, Portugal The State Key Laboratory of Bioreactor Engineering, Key Laboratory for Ultrafine Materials of Ministry of Education, Engineering Research Centre for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, People’s Republic of China § College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China ‡
3.6. Heparin-Based NGs 3.6.1. Self-Assembled Heparin NGs 3.6.2. Chemical-Cross-Linked Heparin NGs 3.6.3. Stimuli-Responsive Heparin NGs 3.6.4. Heparin NGs for Targeted Delivery 3.7. Gelatin-Based NGs 3.7.1. Chemical-Cross-Linked Gelatin NGs 3.7.2. Gelatin NGs for Targeted Delivery 3.8. Other Natural Polymer-Based NGs 3.8.1. Chondroitin Sulfate-Based NGs 3.8.2. Cycloamylose-Based NGs 3.8.3. Glucan-Based NGs 3.8.4. Curdlan-Based NGs 3.8.5. Hydroxypropyl Cellulose-Based NGs 3.8.6. Dextrin-Based NGs 4. Biodegradable Synthetic Polymer-Based NGs for Drug/Nucleic Acid Delivery 4.1. Polypeptide-Based NGs 4.1.1. Chemical-Cross-Linked Polypeptide NGs for Drug Delivery 4.1.2. Stimuli-Responsive Polypeptide NGs 4.2. Poly(ethylene glycol)-Based NGs 4.2.1. Redox-Responsive Poly(ethylene glycol) NGs 4.2.2. Thermoresponsive Poly(ethylene glycol) NGs 4.3. Polyglycerol-Based NGs 4.4. Stimuli-Responsive Polyacrylamide-Based NGs 4.4.1. Redox-Sensitive Polyacrylamide NGs 4.4.2. Enzyme-Sensitive Polyacrylamide NGs 4.4.3. pH- and Thermo-Sensitive Polyacrylamide NGs 5. Release Behavior of Biodegradable NGs 5.1. Physical Encapsulation 5.1.1. Size Effect on Release 5.1.2. External Protecting Structure Effect on Release 5.1.3. Drug Modification Effect on Release 5.1.4. Stimuli-Responsive Delivery Systems 5.1.5. Remotely Controlled Release 5.2. Chemical Conjugation 6. Biodegradability, Biocompatibility, and Therapeutic Efficacy
CONTENTS 1. Introduction 2. General Approaches for the Preparation of Biodegradable Polymer Nanogels (NGs) 2.1. Electrostatic Interaction 2.2. Reverse Miniemulsion 2.3. Desolvation/Coacervation 2.4. Hydrophobic Interaction 2.5. Cross-Linking of Micelles 3. Biodegradable Natural Polymer-Based NGs for Drug/Nucleic Acid Delivery 3.1. Chitosan-Based NGs 3.1.1. Self-Assembled Chitosan NGs 3.1.2. Ionic-Cross-Linked Chitosan NGs 3.1.3. Chemical-Cross-Linked Chitosan NGs 3.1.4. Stimuli-Responsive Chitosan NGs 3.1.5. Chitosan NGs for Targeted Delivery 3.2. Pullulan-Based NGs 3.2.1. Self-Assembled Pullulan NGs 3.2.2. Chemical-Cross-Linked Pullulan NGs 3.2.3. Stimuli-Responsive Pullulan NGs 3.2.4. Pullulan NGs for Targeted Delivery 3.2.5. Encapsulated Pullulan NGs 3.3. Dextran-Based NGs 3.3.1. Ionic Interaction-Induced Dextran NGs 3.3.2. Chemical-Cross-Linked Dextran NGs 3.3.3. Stimuli-Responsive Dextran NGs 3.4. Hyaluronic Acid-Based NGs 3.4.1. Self-Assembled Hyaluronic Acid NGs 3.4.2. Ionic Interaction-Induced Hyaluronic Acid NGs 3.4.3. Hyaluronic Acid NGs for Targeted Delivery 3.5. Alginate-Based NGs 3.5.1. Ionic Interaction-Induced Alginate NGs 3.5.2. Redox-Sensitive Alginate NGs 3.5.3. Alginate NGs for Targeted Delivery © 2015 American Chemical Society
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Received: March 4, 2014 Published: August 11, 2015 8564
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Chemical Reviews 7. Cell Uptake Mechanisms 8. Pharmacokinetics and Biodistribution of NGs 9. Conclusions and Perspectives Author Information Corresponding Authors Notes Biographies Acknowledgments Abbreviations References
Review
delivery systems.27 Many of them are dropped from further development in the pipeline to reach the clinical stage due to insufficient in vivo biodistribution, low plasma stability, rapid plasma clearance, and/or high toxicity.4 Similar to the microenvironment of soft tissue of human body, hydrogels have a 3D interconnected structure, which can absorb a big amount of water. Hydrogels can be designed in the form of continuous macroscopic networks, which can be named as macrohydrogels. They can also be developed as discrete particles. In this case, if the confined dimensions are in microscale (above 1 μm), they are called microgels.28 When the hydrogel particles reach the submicrometer range, they are known as nanogels.28 Therefore, nanogels (NGs) are physically or chemically cross-linked three-dimensional hydrophilic polymer networks with sizes up to a few hundred of nanometers that swell in water.28−30 As in the scientific literature, the term “nanogel” is also used for drug/gene carriers with a size that can reach a few hundred of nanometers; in this Review, we will also apply it when referring to materials in that size range.31−35 NGs have Hamaker constants similar to those of water, and thus own good stability in biological fluids due to the existence of the low driving forces for their aggregation.36 As compared to other nanocarriers, NGs usually have good biocompatibility, high aqueous dispersibility, and well-defined structure.28,29 They also possess easy drug loading ability and multifunctional stimuli-response properties (triggered by pH, temperature, redox, and/or enzyme(s) in the targeted sites).37 These advantages make them ideal systems to load various therapeutic agents through proper physical encapsulation or chemical conjugation.38 Furthermore, their flexibility and softness may allow their easier penetration ability through human skin while maintaining the bioactivity of the therapeutics, when compared to those of the corresponding rigid NPs.39 Their flexibility can also help prolong the circulating lifetime through reducing the possibility of their entrapment by macrophages.40 Moreover, NGs are proven to be more efficiently taken up by cells than conventional nanocarriers such as liposomes, which are less stable than NGs,41 resulting in an improved bioavailability and safety of the therapeutics in vivo.42 NGs are classified into nondegradable and degradable NGs. Among these, biodegradable NGs are more promising for biomedical applications, because they enable a more intelligent delivery of drugs and/or nucleic acids, through manipulating their physical properties or degradability under specific cellular microenvironments. The final biodegraded products may result in a reduced in vivo toxicity in comparison with the nondegradable materials. Biodegradable NGs can also be functionalized with targeting ligands and stimuli-sensitive groups, which can recognize cells/tissues of interest in vivo and suffer cleavage of special bonds triggered by a particular stimulus in the specific microenvironment, respectively, releasing therapeutics in a spatial-temporal way to exert their maximum efficacy.43,44 Their finely controlled architecture and cell-mediated properties make NGs effective nanocarriers for intelligent delivery of drugs/nucleic acids in vivo for treating many diseases, like cancer, neurological disorders, bone degeneration, etc.31,45 In the past few years, overwhelming research has been involved in the development of NGs for the delivery of different kinds of therapeutics. Several review papers have summarized NGs for biomedical applications, but most of them focus on synthetic nonbiodegradable NGs.29,31,45−47 Therefore, in this Review, for the first time, we systematically review the
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1. INTRODUCTION Despite the extensive research on therapeutic delivery, many problems still exist on how to develop a proper platform (a carrier) that can deliver drugs/nucleic acids to specific sites of the body and/or target cells and therefore treat diseases. These problems may be associated with a low efficacy caused by carrier plasma instability and high toxicity, clearance by the reticuloendothelial system (RES), as well as the existence of various intracellular barriers (plasma membrane, endosome entrapment, lysosomal enzymatic degradation, nuclear membrane, etc.).1−5 Nanoparticles (NPs) that are typically defined as particles have at least one dimension at the nanoscale.6,7 NPs are able to improve the efficacy of therapeutic agents (e.g., higher bioactivity and reduced toxicity) by overcoming different obstacles faced by drugs (e.g., low aqueous solubility,8 burst release,9 high toxicity,10 low stability,10 and/or multidrug resistance,11 etc.) and/or by nucleic acids (limited intracellular uptake,12 susceptible to the action of enzymes,13 etc.).14−16 Until the early 1970s, the idea on intravenous administration of therapeutics using particle suspensions was considered impossible because of the likely risks of embolism. However, up to now, nanotechnology has broken this impossibility and is expected to continuously create novel nanomaterials for treatment of various diseases.17 An ideal delivery system should carry therapeutics through the vasculature without drug leakage,18 and then selectively accumulate and exert therapeutic effect on diseased cells or tissues instead of normal ones, thus achieving optimal efficacy while reducing or avoiding undesired side effects.19 As a platform for intracellular delivery of therapeutics inside the human body, nanocarriers need to satisfy several requirements: (a) they should be biocompatible; (b) they should be able to improve the solubility of hydrophobic drugs or effectively encapsulate hydrophilic drugs; (c) they should protect drugs from undesirable interactions with biological milieu components and maintain their stability during circulation period, optimizing pharmacokinetics and biodistribution to reach the diseased tissue; (d) they should recognize the diseased location and selectively accumulate there instead of normal tissues; (e) they should be effectively internalized into the targeted cells (unless a drug acting through cell surface receptors is being applied), and, through intelligent disassembly triggered by stimuli in the intracellular microenvironment, efficiently deliver therapeutics to targeted cellular compartments; and (f) they should biodegrade into small fragments to be completely eliminated by renal clearance without leading to permanent accumulation in normal organs like heart or liver, which can cause adverse effects to the body, as illustrated in Figure 1. Recently, various nanosystems including but not limited to liposomes,20 micelles,21 dendrimers,22,23 hydroxyapatite NPs,24 and nanotubes25,26 have been proposed as drug or nucleic acid 8565
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Figure 1. Schematic illustration of biodegradable NGs as an intracellular therapeutic delivery platform. NGs as an ideal delivery system should effectively encapsulate therapeutics and have an ability to carry them through the vasculature without burst drug leakage during their circulation in the blood. At the same time, they need to maintain a moderate stability, and through passive and active targeting, allow for selective accumulation at the targeted location (e.g., tumor site), with minimum nonspecific accumulation at normal organs, such as the liver, kidneys, spleen, and/or lungs, etc. Later, the nanomedicines should be effectively taken up by the targeting cells. After that, the therapeutics, upon being triggered by internal (e.g., pH, glucose, redox potential, and lysosomal enzymes) or external stimuli (e.g., temperature, magnetic field, ultrasound, and light), will be efficiently released at the targeted location to exert their action. Finally, the nanogels should be degraded into small components to be completely eliminated preferably by renal clearance, without leading to permanent accumulation in normal organs, which may cause adverse effects.
employed to form NGs through ionotropic gelation. For example, anionic tripolyphosphate (TPP) is able to cross-link cationic chitosan (CTS) with a pKa of approximately ∼6.5 to get NGs by ionic bridging of the CTS chains.51−53 The stability of the NGs can be improved by decreasing the CTS concentration and the TPP/CTS ratio, as well as by increasing the ionic strength (ca. 500 mM).54 On the other hand, anionic alginate (ALG) can form reversible NGs by ionic cross-linking with di-/multivalent cations (e.g., Ca2+).55
methods to prepare NGs and the recent progress on the fabrication of biodegradable NGs based on natural and synthetic polymers. Special attention is given to biodegradable NGs in the last 5 years, which have thermo, pH, and/or redoxresponsive properties, as well as targeting ability for drug/ nucleic acid delivery applications. The drug release behavior, the cellular uptake process, and the pharmacokinetics and biodistribution of NGs, which play important roles in the achievement of effective therapeutic efficacy, are highlighted. Current challenges on the future developments of biodegradable NGs are discussed.
2.2. Reverse Miniemulsion
Reverse miniemulsion is another common method employed for development of NGs. In this case, an aqueous solution of hydrophilic precursors is dispersed in an organic solvent in the presence of surfactant to form stable homogeneous droplets. The dispersed phase will be gelated through chemical and physical cross-linking. After removal of the organic solvent, highly monodispersed NGs are obtained. For example, ALG/ Cys NGs with redox-sensitivity are developed by gelating ALG using cystamine (Cys) as a cross-linker via reverse miniemulsion for delivery of an anticancer drug (Figure 3).56 pHresponsive CTS NGs can be formed through in situ precipitation of chitosan upon the addition of ammonia via reverse miniemulsion using Span 40 as surfactant.57 Also, biodegradable synthetic polymer-based NGs are developed by
2. GENERAL APPROACHES FOR THE PREPARATION OF BIODEGRADABLE POLYMER NANOGELS (NGs) 2.1. Electrostatic Interaction
NGs can be formed through complexation between polyelectrolytes with opposite charges in highly diluted aqueous solution through electrostatic interactions.48 The molecular weight (Mw) and/or nonstoichiometric charge ratio of anionic polymer to cationic one are used to adjust the size and surface charge of the formed NGs. Generally, the mixing of oppositely charged polyelectrolytes with equivalent charge ratio tends to induce flocculation, regardless of the Mw of the polymers used (Figure 2).49,50 Multivalent anions or cations can also be 8566
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Figure 2. Formation of colloidal polyelectrolyte (PEC) complexes based on chitosan (light gray) and dextran sulfate (black) through the hydrophobic segregation of complexed segments. Four limit cases are considered according to the charge of the colloid and the molecular weight (Mw) of the components. (a,c) High Mw chitosan versus low Mw dextran sulfate; (b,d) low Mw chitosan versus high Mw dextran sulfate. Adapted with permission from ref 49. Copyright 2010 Elsevier.
be developed using this method. For example, thermosensitive NGs are obtained by grafting N-isopropylacrylamide (NIPAM) blocks onto pullulan or chitosan (Figure 5).64,65 Folic acid (FA) can act as a targeting ligand for folate receptor (FR) overexpressing cancer cells (e.g., several epithelial malignancies, including ovarian, renal, lung, and breast cancers).66 FA has a hydrophobic character and can be conjugated onto hyaluronic acid, which is able to form NGs with enhanced targetability for MCF-7 cells (a human breast cancer cell line).67 However, the NGs obtained by this approach have low stability because they just have a physically cross-linked structure.
radical polymerization of 2-(dimethylamino)ethyl methacrylate by a reverse miniemulsion method.58 2.3. Desolvation/Coacervation
Desolvation/coacervation is a process during which a nonsolvent is added into a homogeneous solution of polymer(s) to induce the formation of nanosized polymer complexes as a dispersed phase. Under this condition, a cross-linker can be introduced to fix the nanocomplexes to form NGs. This method is simple, and the surface of the formed NGs can easily undergo further functionalization. For instance, gelatin (GEL) NGs can be prepared through desolvation/coacervation (water as solvent and acetone as nonsolvent) using glutaraldehyde as cross-linker.59 Cisplatin is a key drug for chemotherapy of gastrointestinal, genitourinary, and lung cancers.60 Cisplatin can be loaded into the GEL NGs via a ligand exchange reaction of Pt(II) from the chloride to the carboxyl group in the NGs, followed by conjugation with biotinylated epidermal growth factor (bEGF, a targeting ligand for EGFR-overexpressing cells). Cisplatin-loaded GEL-bEGF NGs are able to target human A549 lung cancers with improved therapeutic efficacy in vivo (Figure 4).61
2.5. Cross-Linking of Micelles
Because polymeric micelles have instability and uncontrolled drug release behavior, the shell or core of the micelles can undergo further cross-linking to form NGs.68 For instance, diacrylated Pluronic (Plu) micelles are photo-cross-linked into Plu NGs with thermo-sensitivity.69 Using ethylene glycol dimethacrylate as cross-linker, diacrylated poly(lactide)-poly(ethylene glycol)-poly(lactide) (PLA-PEG-PLA) micelles can be cross-linked to form biodegradable PLA-PEG-PLA NGs via radical polymerization (Figure 7j).70 Various thiolated polymer (e.g., alginate, heparin, hydroxypropyl cellulose) micelles are able to form redox-sensitive NGs via cross-linking by oxidation of their free thiol groups (Figure 6).71−73
2.4. Hydrophobic Interaction
Hydrophobic groups can be grafted on hydrophilic polymers to obtain polymeric amphiphiles, which are able to self-assemble into NGs in aqueous solution.62,63 Various functional NGs can 8567
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Figure 3. Schematic illustration of formation, drug loading, and release of alginate/cystamine (ALG/Cys-Dox) NGs via reverse miniemulsion. Scanning electron microscope (SEM) images of (a) ALG/Cys and (b) ALG/Cys-Dox NGs. Dithiothreitol (DTT) is used as a small-molecule redox reagent to mimic reducing intracellular environment. Adapted with permission from ref 56. Copyright 2013 American Chemical Society.
3.1. Chitosan-Based NGs
3. BIODEGRADABLE NATURAL POLYMER-BASED NGs FOR DRUG/NUCLEIC ACID DELIVERY
Chitosan (CTS) is a typical cationic linear polysaccharide, which is obtained by the N-deacetylation of chitin. CTS is composed of randomly distributed β-(1,4)-linked D-glucosamine and N-acetyl-D-glucosamine units (Figure 7a).75 CTS can be used to prepare hydrogels with excellent biocompatibility, enzymatic degradability (in vivo by lysozyme and chitosanase enzymes), low toxicity, and immuno-stimulatory activities.91,92 As one of the hydrophilic natural polymers, CTS can be grafted with various hydrophobic groups to form polymeric amphiphiles, which are able to self-assemble into NGs.62 The cationic nature of CTS under acidic conditions can be used to prepare NGs through polyelectrolyte complexation.93 CTS can also be chemically cross-linked into NGs through the reaction between the amino groups on its backbone and the different cross-linkers.62 CTS-based NGs are able to act as promising platforms for drug,94 protein,93 or gene delivery.50 3.1.1. Self-Assembled Chitosan NGs. CTS can be modified by conjugation with hydrophobic groups to form polymeric amphiphiles, which are able to form CTS NGs via self-assembly. It is a simple method because neither additional cross-linker nor surfactant is required to prepare the NGs.62,63 However, because the amino groups of CTS have a pKa value of ∼6.5, it tends to aggregate under physiological conditions. To increase its physiological stability, CTS can be conjugated with ethylene glycol to obtain glycol chitosan (G-CTS) to increase its water solubility, while retaining its positive charge under physiological conditions.95 After that, G-CTS can be further grafted with hydrophobic deoxycholic acid (DOCA) to form an amphiphilic polymer, which can be self-assembled into G-CTSDOCA NGs (52-222 nm). Exendin-4 (Ex4) is a hormone that can be found in the saliva of the Gila monster and that has a low half-life in bloodstream. To improve this weakness, Ex4 was
On the basis of their origin, biodegradable polymers are classified into two types, that is, biodegradable naturally occurring polymers and biodegradable synthetic polymers.74 Polysaccharides (e.g., chitosan (CTS),75 pullulan (PUL),76 dextran (DEX),49 hyaluronic acid (HA),77 alginate (ALG),78 heparin (HEP),79 and chondroitin sulfate (CS)80), and proteins (e.g., gelatin (GEL)81) belong to the natural biodegradable polymer class. These polymers can be degraded by biological means,82,83 usually have good biocompatibility, and may provide biologic cues,84 influencing cell adhesion, proliferation, and/or differentiation.85,86 However, their uncontrolled structure, degradability, drug release behavior,87 and potential immunogenic responses88 limit their biomedical applications. Synthetic polymers, like polypeptides, polyesters, and polyphosphazenes, etc., have well-controlled structures, which can be employed for the development of different kinds of NGs with controllable degradability, stability, and drug release properties.70 However, synthetic polymers lack biological cues.89 Therefore, intensive investigation has been performed on the integration of natural and synthetic polymers to develop NGs with improved properties for therapeutic applications.90 Chemical structures of some typical natural and synthetic polymers have been summarized in Figure 7. In this Review, biodegradable NGs are divided into natural polymer-based NGs and synthetic polymer-based NGs, according to the matrix used for their fabrication. Partially degradable NGs made of nondegradable synthetic polymers (like poly(ethylene glycol) and polyacrylamide) but having degradable linkages in their formulation are also included in the discussion. 8568
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Figure 4. Schematic representation of formation of cisplatin formulations: (a) gelatin (GEL) NGs are prepared by a desolvation method; (b) cisplatin-loaded GEL NGs are spontaneously formed through a ligand exchange reaction of Pt(II) from the chloride with the carboxyl groups in GEL NGs; and (c) surface modification of GEL NGs is performed with biotinylated epidermal growth factor (bEGF), which displays an in vitro and in vivo targetability with improved anticancer effect on lung cancer. Adapted with permission from ref 61. Copyright 2009 Elsevier.
522 nm and surface potential of 33 mV.96 The cationic nature of CTS also affords the easy formation of complexes with negatively charged DNA. However, the CTS/DNA interaction may be too strong, thereby preventing the dissociation of DNA from CTS NGs upon their internalization into 293T cells (an epithelial human embryonic kidney cell line) and resulting in low gene transfection efficiency, which is less than that treated with commercially available liposome formulations. The incorporation of ALG in the NGs is helpful to adjust the interaction between CTS and DNA, contributing to improved gene transfection in 293T cells (4 times higher than that of CTS NGs without ALG).50 Besides anionic polymers, multivalent anionic small molecules like TPP can be employed to cross-link CTS into CTS/ TPP NGs through inter- and intramolecular electrostatic interaction between the phosphate groups of TPP and the amino groups of CTS (Figure 8).48,93 However, as one of the key factors in the function and shelf life for nanomedicines, the
acylated with palmitic acid to form a peptide derivative (Ex4C16), which was then loaded into G-CTS-DOCA NGs. In vivo studies showed that after pulmonary administration, these NGs remained in the lungs of type 2 diabetic mice for about 72 h. Taking advantage of the hydrophobic interactions between hydrophobic C16 and DOCA, the inhaled Ex4-C16-loaded NGs maintained a higher hypoglycemic duration than Ex4loaded NGs. These results indicate the potential of G-CTSDOCA NGs for the delivery of long-acting inhalation therapeutics for diabetes treatment.62 3.1.2. Ionic-Cross-Linked Chitosan NGs. Generally, ionically cross-linked NGs have better stability than selfassembled NGs with a micelle structure. As a kind of cationic polysaccharide, CTS is able to form NGs with anionic polyelectrolyte through electrostatic interactions.52,53 For example, through the electrostatic interactions between the carboxyl groups of alginate and the amine groups of CTS, CTS/ALG NGs can be formed with a hydrodynamic size of 8569
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Figure 5. CTS-PNIPAM NGs are prepared by grafting N-isopropylacrylamide (NIPAM) onto the chain of chitosan (CTS) via free radical polymerization using N,N-methylene bis(acrylamide) (MBA) as a cross-linker. The formed NGs effectively encapsulate oridonin (ORI) under probe sonication in aqueous solution. Transmission electron microscopy (TEM) images of the unloaded (a) and ORI-loaded (b) NGs. Adapted with permission from ref 65. Copyright 2011 Elsevier.
methotrexate-loaded NGs present a sustained drug release behavior (about 65−70% release during 48 h).99 However, CTS NGs are toxic because of their highly positive-charged surface.100 To improve their biocompatibility, stability, and targetability, cationic CTS/TPP NGs can be further modified with different anionic biopolymers. For example, coating of CTS/TPP pregels with ALG is able to increase their stability under physiological conditions and decrease cytotoxicity as compared to uncoated positively charged CTS NGs.53 ALG can also be incorporated into CTS/TPP systems to form CTS/TPP/ALG NGs (260−525 nm) by the addition of TPP/ALG solution into CTS solution. The NGs can be used to load insulin with an ability to enhance its systemic absorption after nasal administration to conscious rabbits, consequently prolonging the hypoglycemic response of
physical stability of CTS/TPP NGs is not sufficient after 6month storage at 40 °C.97 The stability of CTS/TPP NGs can be affected by the CTS concentration, TPP/CTS ratio, and ionic strength. Generally, a higher CTS concentration and TPP/CTS ratio lead to less stability.54 CTS/TPP NGs are water-swollen colloids, which tend to aggregate via ionic bridging by TPP.51 The NGs are more stable when prepared and stored under saline conditions instead of water, due to the formation of smaller size particle under the former conditions. For instance, the addition of NaCl during the formation of CTS/TPP NGs can dramatically slow the process of aggregation by suppressing the ionic bridging.98 CTS/TPP NGs (59 nm) are able to load an anticancer and autoimmune drug, methotrexate. After coating with polysorbate 80, the 8570
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Figure 6. Alginate is first partially oxidized by sodium periodate (NaIO4), followed by conjugation with hydrophobic 4-amino thiophenol (TSA) to get an amphiphilic polymer (ALG-TSA). ALG-TSA can be self-assembled into nanosized micelles, which are able to be further cross-linked to form ALG-TSA NGs upon oxidation of the thiol groups on ALG-TSA polymer. (a) TEM images of the ALG-TSA NGs. Adapted with permission from ref 71. Copyright 2012 Elsevier.
cross-linker is added to fix the emulsified CTS droplets. After removal of the organic solvent, CTS NGs can be obtained. For instance, CTS/PEG NGs can be formed by cross-linking CTS with polyethylene glycol (PEG) dicarboxylic acid (Mw, 600) via reverse miniemulsion. PEGylation is able to improve the water solubility and stability of the CTS NGs under physiological conditions. The size of CTS/PEG NGs can be tuned from 240 to 470 nm when the pH value changes from pH 7.4 (physiological pH) to pH 2.0 (pH in stomach microenvironment).104,105 3.1.4. Stimuli-Responsive Chitosan NGs. To achieve optimal therapeutic efficacy, it is very important to deliver the therapeutics at the target site (spatial control) and/or at the right time (temporal control). In this case, stimuli-responsive delivery systems are receiving more attention, because drugs and/or nucleic acids can be released in a more controllable way if triggered by internal (e.g., pH, glucose, redox potential, and lysosomal enzymes) or external (e.g., temperature, magnetic field, ultrasound, and light) stimuli.106 It is known that slightly acidic environments are present in solid cancer extracellular environment (pH 6.5−7.2), endosomes (pH 5.0−6.5), and lysosomes (pH 4.5−5.0) as compared to normal tissues with pH 7.4.107,108 Likewise, a higher concentration of glutathione (GSH) tripeptide exists in cytosol, mitochondria, cell nucleus, and some tumor cells than that in the normal extracellular environment.109−111 Therefore, various kinds of stimuliresponsive CTS NGs have been developed for drug delivery applications.106 As a kind of weak polybase, CTS tends to undergo protonation under acidic conditions. Therefore, CTS-based NGs exhibit an acidic-accelerated drug release behavior due to an increase in drug diffusion rate caused by enhanced swelling/ dissolution/degradation or conformational changes of CTS
insulin (up to 5 h).93 Besides the modification with ALG, anionic hyaluronic acid is also utilized for such a coating to obtain CTS/TPP/HA NGs with improved long-term stability and biocompatibility (the IC50 value of the nanogels was determined using macrophages in culture and was shown to increase from 0.7 to 1.0 to 1.8 mg/mL when using the HA coating).100 CTS/TPP/HA NGs can be used for DNA delivery to lower cytotoxicity and increase transgene expression for a longer duration in neural stem cells and organotypic spinal cord slice tissue, as compared to the complexes of PEI and DNA.101 These studies indicate that via a double cross-linking strategy using small molecular polyanionic species and negatively charged polyelectrolytes, the CTS-based NGs can be tuned for therapeutic delivery applications. 3.1.3. Chemical-Cross-Linked Chitosan NGs. As compared to NGs, which are obtained by self-assembly and ionic complexation, chemical-cross-linked NGs have well-defined structure, better stability, and more controllable drug release properties. For example, CTS can react with dialdehyde-capped poly(ethylene glycol) to form G-CTS NGs through a one-step approach via ultrasonic spray in the absence of surfactant. The urokinase (a plasminogen activator)-loaded NGs (200−300 nm) display longer circulation time in vivo than the drug alone. The drug release rate can be accelerated under diagnostic ultrasonic conditions to significantly enhance the thrombolysis of clots, suggesting their potential for treatment of ischemic vascular disease.102 Reverse miniemulsion is another common method to prepare chemical-cross-linked CTS NGs, because emulsion systems are thermodynamically stable and the obtained NGs have monodispersed size distribution, which is controlled by the presence of surfactant.103 In this case, an acidic aqueous solution of CTS is first emulsified in organic solvent in the presence of a surfactant. After that, a chemical 8571
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Figure 7. Chemical structures of typical biodegradable polymers for preparation of NGs. (a) Chitosan, (b) pullulan, (c) dextran, (d) hyaluronic acid, (e) alginate, (f) heparin, (g) chondroitin sulfates, (h) poly[L-aspartic acid-alt-poly(ethylene glycol)] grafted with capryl group, (i) diacrylated copolymer consisting of poly(ethylene glycol) (PEG) and biodegradable poly(ethyl ethylene phosphate) (diacrylated PEEP-PEG-PEEP); and (j) diacrylated poly(lactide)-poly(ethylene glycol)-poly(lactide) (diacrylated PLA-PEG-PLA).
upon pH decrease.92 The pH-sensitive CTS NGs can be obtained by ammonia-induced cross-linking of CTS droplets via reverse miniemulsion. The size of the CTS NGs can be adjusted by changing the Mw of CTS (the Mw increase from 11 to 405 kDa results in a size increase of the CTS NGs from 239 to 464 nm).57 Furthermore, alternative pH-sensitive groups can be grafted onto CTS to enhance its pH sensitivity.38,112,113 For example, hydrophobic 3-diethylaminopropyl (DEAP) groups with a pKb in the range of 7.0−7.3, which is similar to the extracellular pH in solid tumor tissues, can be grafted on glycol chitosan to form self-assembled G-CTS-DEAP NGs (100 nm) at physiological conditions. The NGs are able to load doxorubicin (DOX, a drug used in cancer chemotherapy of some leukemias and Hodgkin’s lymphoma, as well as cancers of the breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple myeloma, etc.).114 The nanosystems enable an acidaccelerated DOX release due to the disintegration of the NGs
through the protonation of both DEAP and CTS under acidic conditions. The DOX-loaded NGs result in an increased DOX uptake in human nonsmall lung carcinoma A549 cells under slightly acidic conditions.113 It has been reported that the intracellular environment has a higher GSH concentration (about 2−10 mM, around 1000 times higher than the extracellular environment (about 2−20 μM)),110 and that some tumor cells have several times higher GSH concentration than normal cells.111 Taking advantage of such properties, various reducible nanocarriers have been developed for therapeutic delivery, which remain stable in the extracellular space in normal tissues, while, once inside cells, they may be easily degraded to release the therapeutics.106 For example, thiolated lactosaminated carboxymethyl chitosan (LAC-CMC-CTS) is able to form NGs in situ through the oxidation of its thiol groups and co-cross-linking by CaCl2. The formed NGs can encapsulate a model drug, glycyrrhizin 8572
DOI: 10.1021/cr500131f Chem. Rev. 2015, 115, 8564−8608
Chemical Reviews
Review
Figure 8. Schematic representation of molecular interactions between chitosan (CTS) and tripolyphosphate (TPP).
glycyrrhizin can be released in the same period in the presence of 1 mM GSH (intracellular-mimic conditions). Through ear vein injection, the drug-loaded NGs present reduced renal excretion rate (in rabbits) and enhanced therapeutic accumulation in the mouse liver.115 In addition, thermosensitive blocks can also be incorporated into CTS NGs to acquire thermoresponsiveness. For example, Pluronic (Plu) polymers consisting of a poly(ethylene oxide)poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) structure can form micelles upon temperature increase. However, the micelle structure is not stable. Concerning this problem, more stable thermosensitive NGs can be prepared through cross-linking of the micelles with diacrylated Plu.69 For instance, CTS can be first acrylated by its reaction with glycidyl methacrylate. After that, photopolymerizing diacrylated Plu and acrylated chitosan (Ac-CTS) in aqueous solution results in the formation of Ac-CTS/Plu NGs (50−150 nm, 10−12 mV). These more stable and biocompatible NGs have higher in vitro cellular uptake ability (in squamous-cell carcinoma (SCC7) of skin cancer) and in vivo tumor accumulation and retention in SCC7 tumor-bearing mice, as compared to Plu NGs.116 The CTS/Plu NGs can be used to encapsulate hydrophilic proteins of various sizes including bovine serum albumin (BSA) (67 kDa) and insulin (6 kDa). The NGs, probably due to their thermo-sensitivity, flexibility, and softness, might be useful to direct the therapeutics to efficiently penetrate human skin while maintaining their bioactivity (Figure 9).39 Because of the protonation nature of CTS under acidic conditions, the thermosensitive NGs containing CTS can also have pHsensitivity. For instance, CTS/PEG NGs (50 mg DOX/100 mg NGs),212 the Ca-ALG/PLL NGs have a less sustained drug release ability in vitro and lower targeting efficiency to LACA mice in vivo than Ca-ALG/CTS NGs.219 Furthermore, Ca-ALG/CTS NGs are useful for delivery of insulin,220 and antisense oligonucleotides (short lengths of single-stranded RNA or DNA that may be used to silence genes).221,222 For example, because the delivery of insulin via the oral route remains a challenging task,223 CaALG/CTS NGs are used in an oral insulin formulation to decrease insulin degradation in the gastric environment. To improve its stability, insulin is first mixed with cationic β-CD polymers (CPβCDs) to form CPβCDs/insulin complexes via electrostatic attraction, which are then loaded into Ca-ALG/ CTS NGs (
