Stimuli-sensitive crosslinked nanoparticles, such as crosslinked micelles and hydrogel nanoparticles, have unique advantages including high stability, high drug loading capacity, and ability to respond to environmental stimuli.170–172 Hydrogel nanoparticles, i.e., nanogels, are swollen nanometer-sized networks comprising hydrophilic or amphiphilic polymers.170, 171 Nanogels may be composed entirely of a polymeric network or a core-shell structure with a hydrogel core or shell.171 Stimuli-sensitive nanogels with an hydrophilic and biocompatible shell, such as stimuli-dependent swellable crosslinked micelles, have received much attention due to their smart swelling–deswelling transitions and improved biocompatibility. Drugs can be loaded in the temporarily stable nanocarriers and the release of the drugs can be triggered by environmental stimuli at desirable sites. In the past decade, polypeptide-based crosslinked nanoparticles, such as crosslinked micelles and nanogels, which respond to external stimuli, such as pH, reducing environment, and dual stimuli, have been developed for different drug delivery applications. These systems have been fabricated through various methods including crosslinking of preformed polymer chains or self-assembled nanoaggregates, precipitation polymerization, and one-step ring-opening polymerization.
3.3.1. Reduction-Sensitive Crosslinked Nanoparticles
Nanoparticles crosslinked by covalent bonds capable of responding to intracellular microenvironment, in particular the acidic endosomal pH and the reducing environment of the cytoplasm, are interesting for intracellular drug delivery.5, 17 The development of reduction-sensitive nanocarriers is commonly achieved by incorporation of disulfide bonds to the drug delivery systems, due to selective cleavage of the disulfide bonds in the cytoplasm. The disulfide bonds are stable in the relative oxidation environment in vivo, such as body fluids and extracellular space, resulted from a low concentration (2–20 μM) of glutathione (GSH).17 In contrast, the disulfide bonds can be cleaved in the relative reducing environment of the cytoplasm due to a relatively high GSH concentration (0.5–10 mM). Consequently, disulfide-crosslinked nanoparticles based on polypeptides have also been developed for intracellular drug and gene delivery.173–175
Reversible shell crosslinked micelles based on a series of poly(L-cysteine)-b-PLLA diblock copolymers (PLCys-b-PLLA, Scheme4a) have been synthesized.173 PLCys-b-PLLA was synthesized via ROP of β-benzyloxycarbonyl-L-cysteine N-carboxyanhydride (ZLC-NCA) by using amino-terminated PLLA as a macro-initiator, followed by deprotection of the benzyloxycarbonyl groups using HBr. Micelles with a PLLA core and a PLCys shell were firstly formed in the presence of DTT, and then shell crosslinked micelles containing disulfide bonds were obtained by removing DTT and aerial oxidation. Ellman's assay suggested that less than 6% free thiols remained after oxidation. The decrosslinking of the micelles was achieved by addition of DTT. DLS and ESEM measurements revealed that the particle size increased slightly from 41.7 nm to 55.1 nm after adding DTT, and decreased to 47.1 nm again following removal of DTT, suggesting a reversible crosslinking–decrosslinking process of the micelles. Additionally, no intermicellar crosslinking was observed by DLS and ESEM tests. A hydrophobic model drug, rifampicin, was loaded into the shell crosslinked micelles by self-assembly of PLCys-b-PLLA in the presence of rifampicin, followed by aerial oxidation.176 The drug-loading content and loading efficiency were 15.0% and 17.5, respectively. The drug-loaded crosslinked micelles showed a size (∼65 nm) slightly higher than the particles without drug. A faster release profile was observed in the presence of DTT, due to decrosslinking of the particles caused by cleavage of the disulfide bonds by DTT. In addition, a model protein containing a free cysteine residue, i.e.; BSA, was successfully conjugated to the diblock copolymer via oxidation, and the release of BSA from the copolymer was triggered by addition of reducing agent.
Scheme 4. Chemical structures of some representative polypeptide-based block copolymers and crosslinkers that are used for the preparation of crosslinked micelles and nanogels.
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Reversible-core crosslinked micelles containing disulfide bonds have also been developed. Kataoka and co-workers have prepared a series of core-crosslinked polyion complex (PIC) micelles via disulfide bonds through mixing of thiolated PEG-b-PLLys and PAsp, followed by crosslinking of the micellar core via aerial oxidation.174 The thiolation of PEG-PLL was performed via the coupling reaction between PEG-b-PLLys and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane (IM).177 The IM-conjugated PLLys block has a higher charge density than the SPDP-modified PLLys block, due to the introduction of positively charged imino moieties. The chemical structure of a thiolated PEG-b-PLLys (denoted as PEG-b-P(LLys-MP)) obtained by treating the SPDP-modified PEG-b-PLLys using DTT is shown in Scheme 4b. The core-crosslinked PIC micelles showed an almost constant diameter with the salt concentration ranging from 0 to 0.5 M, in contrast to a significant decrease in stability of the parent uncrosslinked PIC micelles.174 On the other hand, the disulfide crosslinked micelles were found to be dissociated rapidly in the presence of a reducing agent, DTT, indicating a reduction-induced cleavage of the disulfide bonds. It is noteworthy that the reductive stability of disulfide bonds can be affected by the substitution groups close to the disulfide bonds.178 In subsequent studies, disulfide core-crosslinked PIC micelles based on thiolated PEG-PLLys and negatively charged biopharmaceuticals, including antisense oligonucleotide (ODN), pDNA, and siRNA, were developed.177, 179, 180 It was found that the core-crosslinked PIC micelles with thiolation degrees of 10 and 26% displayed sizes comparable to that of the uncrosslinked micelles.179 The core-crosslinked PIC micelles exhibited a high stability in the presence of a competing polyanion, i.e., poly(vinyl sulfate) (PVS), and showed a markedly enhanced resistance to nuclease compared to free ODN and uncrosslinked micelles. The release of loaded biopharmaceuticals from the crosslinked PIC micelles was triggered by addition of reducing agents, such as GSH. A higher GSH concentration and a lower crosslinking density resulted in a faster release rate. The disulfide crosslinked PIC micelles containing pDNA with a size of ∼100 nm showed enhanced in vitro transfection efficiency than uncrosslinked micelles.177 This was believed to be due to a higher stability of the crosslinked micelles in culture medium, leading to an increased cellular uptake.181 In addition, the gene transfection efficiency was also influenced by a balance between the charge density and thiolation ratio. A freeze-dried formulation of the core-crosslinked PIC micelles containing pDNA was subsequently developed.182 The micelles with a thiolation degree higher than 13% retained the original size and shape and showed comparable gene transfection efficiency after a freeze-drying/reconstitution process. After intravenous injection of the crosslinked micelles with a thiolation degree of 37% into mice via the orbital vein, a gene expression was observed in the liver, compared to no gene expression detected for the group treated with naked pDNA. Crosslinked PIC micelles conjugated with a targeting ligand, i.e., cRGDfk, have also been prepared by thiolated cRGDfk-PEG-PLLys and pDNA.181 The introduction of cRGDfk ligand led to an increase in transfection efficiency against HeLa cells expressing αvβ3 integrin receptors. Based on CLSM observation, the increase in transfection efficiency was proposed to be due to cellular internalization through caveolae-mediated endocytosis. Additionally, a series of disulfide crosslinked PIC nanoparticles based on oligopeptides and glycopeptides containing both LLys and cysteine residues have been developed by Rice and co-workers.178, 183, 184
Interestingly, disulfide core-crosslinked nanoparticles (CCLNPs) based on polypeptides have been developed by a simple one-step method using a difunctional NCA comonomer. In two separate studies, a difuctional NCA containing a disulfide bond and two NCA rings, i.e., L-cystine-NCA (Scheme 4c), was synthesized.185, 186 Accordingly, disulfide CCLNPs based on PEG and polypeptide was fabricated by one-step ring opening copolymerization of L-cystine-NCA with LPhe-NCA or BLG-NCA using amino-terminated mPEG as macro-initiators. 1H NMR tests indicated that the typical signals of polypeptides were suppressed, suggesting a core–shell structure with a disulfide crosslinked polypeptide core and a PEG shell.185, 186 As shown in Figure8, transmission electron microsopy (TEM) observation revealed that the PEG-poly(LPhe-co-L-cystine) CCLNPs exhibited a uniform spherical morphology with sizes ranging from 50–150 nm, which are smaller than those determined by DLS measurements, likely due to the shrinkage of the nanoparticles during the sample drying process.185 The size of the CCLNPs increased with increasing the overall polypeptide fraction or the content of L-cystine residues (Figure 8A). The CCLNPs remained stable in PBS (pH 7.4) even at an extremely low concentration (1.53 × 10−5 mg mL−1). In contrast, the disulfide crosslinking bonds were cleaved in the presence of 10 mM GSH, rendering an obvious increase in the size of the nanoparticles. Dox-loaded CCLNPs showed a reduction-dependent drug release behavior, as shown in Figure 8B. In PBS without GSH, less than 20% of loaded Dox was released from the CCLNPs at 93.5 h. In contrast, the release was markedly accelerated by addition of GSH, and over 90% Dox was released at 93.5 h in the presence of 10 mM GSH. In addition, an increase in the content of the L-cystine crosslinks or an increase in the overall polypeptide fraction led to a decrease in drug release rate, resulted from an increase in crosslinking density and the formation of a more compact nano-structure. The subsequent CLSM observation revealed effective intracellular delivery of Dox by the Dox-loaded CCLNPs. Strong intracellular fluorescence of Dox was observed within the HeLa cells cultured with the Dox-loaded CCLNPs for 2 h, suggesting intracellular release of Dox from the CCLNPs triggered by GSH-mediated cleavage of the disulfide crosslinks. MTT assays revealed that the polypeptide-based CCLNPs exhibited no significant cytotoxicity and the Dox-loaded CCLNPs showed lower cytotoxicity than free Dox. In addition, a similar GSH-accelerated release of indometacin from a PEG-poly(BLG-co-L-cystine) CCLNP was observed.186 Because the PEG-polypeptide CCLNPs that are prepared through facile one-step ROP of NCAs exhibit an intelligent drug release pattern in response to intracellular reducing environment, it is envisioned that these materials may have potential applications in intracellular drug delivery.
Figure 8. A) TEM image and the hydrodynamic radii (Rh) of the PEG-poly(LPhe- co -L-cystine) CCLNPs: a) CCLNPs-1 (1/2/19, mPEG/LCys/LPhe (molar ratio)), b) CCLNPs-2 (1/6/23), and c) CCLNPs-3 (1/9/32). B) In vitro release of Dox from the Dox-loaded CCLNPs in PBS at pH 7.4 and 37 °C: traces (a), (b) and (c) refer to the release results of CCLNPs-1, CCLNPs-2 and CCLNPs-3, respectively, without the presence of GSH; traces (d) and (e) represent the results of CCLNPs-3 with the presence of 2.5 mM and 5 mM GSH, respectively; traces (f–h) refer to the results of CCLNPs-1, CCLNPs-2 and CCLNPs-3, respectively, in the presence of 10 mM GSH. Reproduced with permission.185 Copyright 2011, Royal Society of Chemistry.
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In addition, an interlayer-crosslinked micelle has also been reported.187 A PEG-b-PAsp(MEA)-b-PAsp(DIP) triblock copolymer was synthesized by Cu(I)-catalyzed alkyne-azide “click” reaction between azido-terminated PEG-b-poly(N-(2-mercaptoethyl) aspartamide) (PEG-b-PAsp(MEA) and alkyne-functionalized poly(N-(2-(diisopropylamino)ethyl) aspartamide) (PAsp(DIP). At pH 10, the triblock copolymer self-assembled into micelles with a three-layer structure including a PAsp(DIP) core, a PEG outer shell and a PAsp(MEA) intermediated layer (Figure9A-a). Due to the effect of aerial oxidation, disulfide crosslinking bonds were further formed within the PAsp(MEA) interlayer, leading to the formation of interlayer crosslinked micelles. Because of the presence of both tertiary amino groups in the core and disulfide crosslinks in the interlayer, the micelles showed pH- and reduction-sensitive behaviors. At pH 5.0 without adding DTT, an interesting nanocage structure was observed (Figure 9A-b). The nanocages showed a markedly enhanced size than the crosslinked micelles, due to complete dissolution of the inner PAsp(DIP) core that is constrained by the crosslinked interlayer. In addition, with addition of DTT (10 mM) at pH 7.4, highly “swollen” micelles were observed due to cleavage of the disulfide crosslinks and the swelling of partially dehydrated PAsp(DIP) segments (Figure 9A-c). Notably, the nanoparticles disassembled at pH 5.0 with the presence of 10 mM DTT (Figure 9A-d). In vitro drug release measurements revealed that Dox-loaded interlayer crosslinked micelles retained stable in PBS at pH 7.4 without DTT (Figure 9B). In contrast, the release of Dox was significantly accelerated by either reducing the pH to 5.0 or addition of DTT (10 mM), and highest release rate was observed in the presence of dual stimuli. CLSM observation demonstrated that fast accumulation of Dox in the nuclei of Bel-7402 cells was detected for free Dox and the Dox-loaded crosslinked micelles, compared to the distribution of Dox mainly in the cytoplasm for PEG-b-PCL micelles (Figure 9C), indicating an enhanced endosomal/lysosomal escape of Dox for the Dox-loaded crosslinked micelles. Further in vivo studies revealed that the Dox-loaded crosslinked micelles showed higher antitumor efficacy than either free Dox or the Dox-loaded PEG-b-PCL micelles.
Figure 9. A) TEM images of the nanoassembly at pH values of a) 7.4, b) 5.0, c) 7.4 with addition of DTT, and d) 5.0 with addition of DTT. The interlayer crosslinked micelles shown in (a) were decorated with Au. In TEM measurements, the Au-decorated crosslinked micelles were not stained and other samples were stained with uranyl acetate. The arrows in (b) indicate the “watermark” of staining agent formed as a result of nanocage shrinkage in sample drying. DTT concentration (if added): 10 mM. B) Quantitative Dox release from the dual-sensitive crosslinked micelles (mean ± standard deviation (SD), n = 3). C) Intracellular Dox release and migration into nuclei observed by confocal laser scanning microscopy (CLSM). Bel-7402 cells were incubated (37 °C) for 6 h at a Dox-equivalent dosage of 10 μg per dish. Dox loading contents: 10.5% in the interlayer crosslinked micelles and 5.1% in PEG3k-PCL3k micelles. Nuclei were stained with Hoechst 33342. Reproduced with permission.187
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3.3.2. pH-Sensitive Crosslinked Nanogels
Nanometer-sized polypeptide networks that undergo swelling–deswelling transitions in response to pH change, i.e., pH-sensitive nanogels,170 are interesting for pH-triggered drug-delivery systems. PEG-b-PAsp nanogels were synthesized by crosslinking of the PAsp blocks using 1,6-hexanediamine (HDA) and N,N′-diisopropylcarbodiimide (DIC) as a crosslinker and a coupling agent, respectively.188 A core–shell structure with a crosslinked PAsp hydrogel core and a PEG shell was formed during the crosslinking. The nanogels exhibited a pH-dependent swelling–deswelling transition, as schematically illustrated in Figure10. As the pH increase from 4 to 9, the size of the nanogels increased from below 20 nm to above 40 nm, attributed to the swelling of the polypeptide core caused by the gradual ionization of the Asp residues at pH above its pKa (∼3.9).4 The nanogels showed a constant diameter in 0.15 M PBS (pH 7.4) as the polymer solution was diluted from 5 to 0.2 mg mL−1, compared to the dissociation of PEG-b-PAsp/Ca2+ ion complex micelles at concentrations less than 1 mg mL−1. Dox-loaded nanogels were obtained by mixing Dox with nanogels in deionized water. A relatively high drug loading capacity (26.6 wt%) was obtained, likely due to the electrostatic interactions between oppositely charged Asp residues and Dox. The loading of drug showed no obvious influence on the particle size. It was found that Dox was rapidly released from the nanogels at both pH 5.0 and 7.4, and a slightly faster drug release profile was observed at pH 5.0 than at pH 7.4. It is noteworthy that the nanogels are in a swollen state at 7.4, which may facilitate drug diffusion. The faster release pattern of Dox at acidic pH was assumed to be attributed to an increased solubility of Dox (pKa = 8.25) and reduced interactions between Asp and Dox.
Figure 10. Schematic illustration of the pH-dependent swelling–deswelling transition of a core-crosslinked PEG-b-PAsp nanogel.
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On the other hand, it was found that the change in the water-solubility of Dox with decreasing pH showed no significant influence on the drug release behavior of a PEG-b-PAsp-b-PLPhe ternary copolymer nanogel.189 PEG-b-PAsp-b-PLPhe triblock copolymers were synthesized via successive ROP of BLAsp-NCA and LPhe-NCA using amino-terminated mPEG as a macro-initiator. Reversible or irreversible nanogels were then prepared by crosslinking the micelles of PEG-b-PAsp-b-PLPhe using an acid-labile ketal-containing crosslinker (Scheme 4d) or a nondegradable crosslinker. The Dox-loaded nanogels containing nondegradable crosslinks exhibited similar drug release profiles at pH 7.4 and 5.0. In contrast, the nanogels crosslinked by acid-cleavable crosslinks showed a markedly enhanced drug release rate as the pH was reduced from 7.4 to 5.0. Hence, an acid-catalyzed hydrolysis of the ketal-containing crosslinks was proposed to be responsible for the accelerated drug release at acidic pH.189 When cultured with MCF-7 cells, the Dox-loaded nanogels with ketal crosslinks resulted in a higher Dox fluorescence intensity within the nuclei than the Dox-loaded nanogels with non-biodegradable crosslinks, indicating an enhanced Dox release from the former nanogels at acidic endosomal/lysosomal environment. Ketal linkages have also been incorporated to the side chains of a polypeptide diblock copolymer, i.e., PEG-b-poly(ketalized serine) (PEG-b-PkSer, Scheme 4e), to fabricate a PEG-b-PLLys analogue with acid-cleavable side chains.190 After crosslinking of PEG-b-PkSer/DNA polyplexes by bis(sulfosuccinimidyl)suberate, crosslinked PIC micelles with both acid-cleavable crosslinks and side chains were obtained. The resulting crosslinked micelles exhibited increased transfection efficiency in the presence of serum than either uncrosslinked PEG-b-PLLys/DNA polyplexes or PEI/DNA polyplexes. CLSM observation revealed an improved dissociation of PEG-b-PkSer and DNA in the cytoplasm compared to the uncrosslinked PEG-b-PLLys/DNA polyplexes, implying an acid-triggered cleavage of the ketal linkages.
Besides the chemical crosslinking methods, photocrosslinking is another commonly used method for in situ crosslinking, for the crosslinking can be performed in a controllable manner with or without the presence of photo-initiators. Chen and co-workers have reported a series of pH-sensitive polypeptide nanogels developed through photo-crosslinking. Photocrosslinkable PEG-b-poly(LGlu-co-γ-cinnamyl L-glutamate) (PEG-b-P(LGlu-co-CLG)) diblock copolymers were synthesized by ROP of BLG-NCA using amino-terminated mPEG as an initiator, followed by grafting cinnamyl alcohol to the LGlu residues.191 PEG-b-P(LGlu-co-CLG) self-assembled into nanoparticles in aqueous solution due to the hydrophobic interactions between CLG segments. Under UV irradiation at 254 nm, the pendant cinnamyl groups within the polypeptide block underwent dimerization, resulting in in situ crosslinking of the polypeptides segments and hence the formation of nanogels. The nanogels showed hydrodynamic radii ranging from 80–135 nm at pH 7.4, depending on the polypeptide block length and the substitution degree of cinnamyl groups. As the pH increased from 4.0 to 7.4, the nanogels exhibited a significant increase in size, due to the swelling of the polypeptide cores caused by gradual ionization of the LGlu residues. MTT assays indicated that both the block copolymers and the nanogels showed no detectable cytotoxicity at concentrations up to 0.1 mg mL−1. Drug-loaded nanogels were prepared by mixing PEG-b-P(LGlu-co-CLG) with a model drug, rifampicin, in aqueous solution, followed by in situ photocrosslinking. The drug-loaded nanogels showed pH-dependent release profiles in vitro. A fast drug release pattern was observed at pH 7.4, compared to only small amount of drug released at pH 4.0, due to the swelling of the nanogels at higher pH.
3.3.3. pH- and Temperature-Sensitive Nanogels
In addition to the nanogels that respond to single stimulus, polypeptide-based nanogels that exhibit swelling–deswelling transitions in response to dual stimuli, such as pH and temperature, have also been investigated for controlled drug delivery. Nanogels based on PNIPAM and PLGlu were synthesized via free radical polymerization of 2-hydroxyethyl methacrylate (HEMA) and PNIPAM grafted PLGlu (PLGlu-g-(HEMA/PNIPAM)) by Chen and co-workers.192 PLGlu-g-(HEMA/PNIPAM) was first prepared by means of conjugating amino-terminated PNIPAM and HEMA to the PLGlu side chains. The nanogels were then obtained by increasing the temperature from 25 °C to 60 °C at pH 8 to form a dispersion of nanoparticles, followed by free radical polymerization of the HEMA residues using ammonium peroxydisulfide (APS) as an initiator. The polymerization of the HEMA residues led to a sharp decrease in the hydrodynamic diameter of the aggregates from 262 nm to about 60 nm, due to the formation of crosslinked nanogels with a more compact structure. The nanogels exhibited pH- and temperature-dependent swelling–deswelling behaviors. At 27 °C, the nanogels showed a decrease in size from ∼70 nm to ∼60 nm as the pH was reduced from 10.0 to 6.0, caused by the gradual protonation of the LGlu segments. Notably, as the pH was further decreased to below 5.5, the particle size increased markedly and large aggregates were detected due to the hydrophobic aggregation of the PLGlu segments. In addition, at pH 7.0, the nanogel size showed a sharp decrease as the temperature was increased to above 36 °C, attributed to hydration-dehydration transitions of the PNIPAM segments. PLGlu/PNIPAM hybrid microgels have also been prepared via free radical copolymerization of HEMA-grafted PLGlu and N-isopropylacylamide (NIPAM) in 0.05 M PBS (pH 7.0) at 60 °C by using APS as an initiator.193 In comparison with the above PLG-g-(HEMA/PNIPAM) nanogels, the microgels exhibited significantly higher size (570 nm) at pH 7.0 and 25 °C. The microgels displayed pH- and temperature-sensitive swelling–deswelling behaviors similar to the nanogels. Because of the dual-stimuli responsive swelling behaviors of the nano-sized and micrometer-sized hydrogel particles, it is envisioned that these materials may be interesting for drug delivery systems, such as oral drug delivery systems.