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Facile fabrication of poly(acrylic acid) coated chitosan nanoparticles with improved stability in biological environments
Accepted Manuscript Facile fabrication of poly(acrylic acid) coated chitosan nanoparticles with improved stability in biological environments Yukun Wu, Jing Wu, Jing Cao, Yajie Zhang, Zhe Xu, Xiuyi Qin, Wei Wang, Zhi Yuan PII: DOI: Reference:
S0939-6411(16)30845-1 http://dx.doi.org/10.1016/j.ejpb.2016.11.020 EJPB 12355
To appear in:
European Journal of Pharmaceutics and Biopharmaceutics
Received Date: Revised Date: Accepted Date:
12 June 2016 17 November 2016 20 November 2016
Please cite this article as: Y. Wu, J. Wu, J. Cao, Y. Zhang, Z. Xu, X. Qin, W. Wang, Z. Yuan, Facile fabrication of poly(acrylic acid) coated chitosan nanoparticles with improved stability in biological environments, European Journal of Pharmaceutics and Biopharmaceutics (2016), doi: http://dx.doi.org/10.1016/j.ejpb.2016.11.020
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Facile fabrication of poly(acrylic acid) coated chitosan nanoparticles with improved stability in biological environments Yukun Wu,† Jing Wu,† Jing Cao,† Yajie Zhang,† Zhe Xu,† Xiuyi Qin,† Wei Wang,† Zhi Yuan*,†,‡ †
Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, and
‡
Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China * Corresponding author E-mail address: [email protected] Tel.: +86-22-23501164 Fax: +86-22-23503510 Abstract Chitosan is one of the most important and commonly used natural polysaccharides in drug delivery for its biocompatible and biodegradable properties. However, poor blood circulation of the chitosan nanoparticles due to their cationic nature is one of the major bottlenecks of chitosan-based drug delivery systems. To address this problem, a versatile platform based on poly(acrylic acid) (PAA) coated ionically cross-linked chitosan/tripolyphosphate nanoparticles (CTS/TPP-PAA NPs), is reported. The zeta potentials of CTS/TPP and CTS/TPP-PAA NPs are approximately 33 mV and -25 mV, respectively. CTS/TPP NPs quickly aggregate in PBS (phosphate buffered saline) and DMEM (Dulbecco's modified Eagle's medium). Conversely, CTS/TPPPAA NPs exhibit excellent colloidal stability in plasma solution for more than 24 h. The PAA coating also endows CTS/TPP-PAA NPs with decreased protein adsorption capacity and improved buffering capacity. More importantly, the residual carboxyl and amino groups on CTS/TPP-PAA NPs provide abundant reactive sites for further functional modifications. Therefore, the CTS/TPP-PAA NPs reported here may be useful as an alternative drug delivery system.
Keywords Chitosan, poly(acrylic acid), surface modification, plasma stability, endosomal escape, drug delivery Introduction Chitosan has been widely utilized for a variety of medical applications, such as cell imaging and drug delivery systems for peptides, genes and low-molecular-weight drugs.[1] As the only known naturally occurring polycationic polysaccharide, chitosan has versatile characteristics, such as bioavailability, biocompatibility, biodegradability, low immunogenicity, and high charge density.[2] Chitosan nanoparticles can be prepared by chitosan-drug conjugates, ionically or covalently cross-linked chitosan, or amphiphilic chitosan derivatives.[3– 10] Among them, ionic cross-linking of chitosan using tripolyphosphate (TPP) as the crosslinker has numerous advantages, such as mild, convenient, non-toxic and organic solvent free preparation conditions.[3,11,12] Despite their biocompatibility and biodegradability, chitosan nanoparticles, like most of the positively charged nanoparticles, always interact strongly with serum components, which causes severe aggregation and rapid clearance from circulation and limits their in vivo application.[13,14] Therefore, tailoring the chitosan nanoparticles towards greater stability in biological environments is important for tumor targeting and
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intracellular drug delivery. The most frequently used method to overcome this drawback is hydrophilic modification of chitosan, using polymers and molecules such as β-cyclodextrin,[15] PEG,[7] and succinic anhydride,[4,6] which enhance chitosan’s solubility in slightly acid and neutral media and confer stability to the chitosan nanoparticles. However, hydrophilic modification of the chitosan backbone can compromise the hydrophobic and cationic integrity of the chitosan core, which can lead to a reduced hydrophobic drug loading capacity[16,17]. At the same time, methods of stabilizing chitosan nanoparticles against aggregation through surface decoration with hydrophilic polymers or macromolecules have rarely been reported.[18] In the present work, we aimed to develop a facile and versatile approach towards the formation of chitosan nanoparticles that were stable in biological media. (Figure 1) First, TPP cross-linked chitosan (CTS/TPP) nanoparticles were fabricated by the standard ionic gelation technique. Second, polyanion poly(acrylic acid) (PAA) was attached to the surface of chitosan nanoparticles through an electrostatic interaction between positively charged protonated chitosan chains and the negatively charged PAA polymer. Finally, the PAA polymer was fixed onto the surface of the CTS/TPP core using water-soluble carbodiimide (EDC) at room temperature. This chitosan-based nanoparticle has some interesting features: (1) the residual carboxyl and amino groups on PAA-CTS/TPP NPs provide abundant reactive sites for further functional modifications. (2) The post-modification of CTS/TPP NPs did not impair the integrity of the chitosan core, which is beneficial for increasing the drug loading capacity and gene condensing capacity. (3) The CTS/TPP-PAA NPs exhibited excellent colloidal stability in PBS and plasma solution. (4) The electrostatic modification of CTS/TPP nanoparticles is simple and versatile, and could easily be applied to a variety of chitosan nanoparticles and analogous nanoparticles.
Figure 1. Scheme for preparation procedure of chitosan-based nanoparticles. . Experimental 2.1 Materials Chitosan was purchased from TCI Co. Ltd. (Japan), with deacetylation degrees of 75.0 to 85.0 %. FITC-labeled chitosan (FITC-CTS) was synthesized according to our previous studies.[10] Poly(acrylic acid) (PAA, molecular weight of 1800 Da), folate (folic acid), sodium tripolyphosphate (TPP), fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) and 1-dimethylaminopropyl-ethylcarbodiimide hydrochloride (EDC) were all purchased from Aladdin (Shanghai, China) and used as received. The other chemical reagents used in this study were all commercially acquired from Tianjin Chemical Reagent Company (Tianjin, China) with analytical reagent grade.
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The human liver carcinoma cell line (HepG2) was purchased from the Shanghai Institute of Biochemistry and Cell Biology cell bank. DMEM medium, FBS, trypsin and other biological reagents were all purchased from DingGuo Biotech. Co. (Shanghai, China). 2.2 Fabrication of chitosan nanoparticles Fabrication of PAA coated chitosan nanoparticles were achieved in three steps, as illustrated in Figure 1. TPP cross-linked chitosan nanoparticles (CTS/TPP NPs) were prepared based on the ionic gelation interaction between positively charged chitosan and negatively charged TPP according to a previously reported method.[19–22] Briefly, chitosan was dissolved overnight in aqueous acetic acid (1 wt %) at a concentration of 1 mg/mL, and the pH was adjusted to 6.0 using sodium hydroxide solution. Under intensive stirring, TPP solution (1 mg/ml) was added dropwise (1 drop per 1-2 s) to the CTS solution in an ice water bath. The solution was stirred for 24 h at room temperature before use. Nanoparticles were ultra-centrifuged at 15,000 rpm at 4 oC for 1.5 h in an ultracentrifuge (Sigma 3K30, UK) and redispersed using a probe ultrasonic processor (Scientz-IID, Ningbo Scientz Biotech., Ningbo, China) with 10W output power, 25KHz ultrasonic frequency for 5 min. Poly(acrylic acid) coated chitosan nanoparticles (CTS/TPP/PAA NPs) were prepared based on an electrostatic interaction between chitosan and poly(acrylic acid), according to the layer-by-layer (LBL) method.[23] Briefly, CTS/TPP NPs were ultra-centrifuged at 15,000 rpm at 4 oC for 1.5 h in an ultracentrifuge (Sigma 3K30, UK) and redispersed in an aqueous solution of 10mg/mL PAA (molecular weight 1800 Da, pH=6.0) using the same procedure mentioned above, during which the PAA was electrostatically attached to the CTS/TPP NPs. The nanoparticles were then separated from the PAA solution and redispersed in MilliQ water (Millipore Milli-Q purification system, MA, USA) using the same procedure mentioned above. Poly(acrylic acid) conjugated chitosan nanoparticles (CTS/TPP-PAA NPs) were prepared by conjugation between the PAA coating and CTS/TPP core using a water-soluble carbodiimide (EDC) as the cross-linker, according to a previously described method.[24,25] Briefly, an EDC aqueous solution (10 mg/mL) was mixed with the CTS/TPP/PAA NPs (1 mg/mL) with sonication and reacted at room temperature overnight to fix the PAA coating via the amide bond between the carboxyl group of PAA and the amino group on the chitosan nanoparticles surface. Subsequently, nanoparticles were collected by centrifugation at 15,000 rpm for 1.5 h. The supernatants were discarded and the pellet was redispersed in MilliQ water. 2.3 Evaluation of nanoparticles stability 2.3.1 Size measurement of nanoparticles The size, zeta potential of nanoparticles were determined using a Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK) equipped with 4 mW He Ne laser at a scattering angle of 90o at 532 nm at 25 C. Polydispersity index (PDI) was also measured to determine particle size distribution. The typical nanoparticle concentration used was 0.1mg/mL in MilliQ water or PBS. The solutions were filtered using a 0.45 micron filter prior to analysis. All the measurements were repeated three times, and an average value was reported. The stability of nanoparticles under simulated physiological conditions was monitored by size measurements at different time points. Chitosan nanoparticles were ultra-centrifuged at 15,000 rpm for 1.5 h, and the obtained pellets were redispersed in MilliQ water, PBS (pH 7.4, 10mM, I=150 mM) and DMEM cell culture medium with 10% FBS, respectively. The nanoparticles were incubated at 37 C under agitation (100 rpm).
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The size and morphology of nanoparticles were measured by transmission electron microscopy (TEM) on a Tecnai G2 F20 S-Twin electron microscopy center (FEI Company, America). To prepare the TEM samples, the sample solution was dropped onto a carbon-coated copper grid, stained with 2% (w/v) phosphotungstic acid aqueous solution, and dried slowly in air. The acceleration voltage was 100 kV. 2.3.2 BSA absorption assay The interaction between bovine serum albumin (BSA) and the chitosan nanoparticles was investigated according to the literature procedure.[26] Approximately 0.4 mL of a dispersion of CTS/TPP and CTS/TPP-PAA nanoparticles (0.5 mg/mL) was suspended in 0.2 mL of aqueous solution with FITC-BSA at an initial concentration of 0.05 mg/mL. The samples were then shaken for 1.5 h at 37 C. Nanoparticles were separated from unbound FITC-BSA by ultracentrifugation at 15,000 rpm and 4 C for 1.5 h. The fluorescence intensity of the supernatant was measured using a fluorescence spectrometer (F-7000, Hitachi, Japan). A solution of 167 μg mL-1 FITC-BSA in MilliQ water was utilized as a control. Samples were excited at a fixed wavelength of 488 nm and spectra were recorded in a wavelength range between 500 and 550 nm. The input/output slit was 5 nm. The PMT voltage was 700V. 2.3.3 Effect of freeze-drying on the stability of chitosan nanoparticles CTS/TPP and CTS/TPP-PAA NPs (1 mg/mL) with or without the commercial cryoprotectant mannitol (1%) were freeze-dried using a Scientz-10N lyophilizer (Xin Zhi company, China). Solutions were firstly frozen to 20 °C for 5 hours. Frozen solutions were then desiccated under vacuum (-0.1MPa) with a primary drying stage at -20°C for 12 hours, and a secondary drying stage at room temperature for 24 hours. The freeze-dried samples were finally redispersed in MilliQ water using a probe ultrasonic processor (Scientz-IID, Ningbo Scientz Biotech., Ningbo, China). And the nanoparticle size and size distribution were measured by the DLS instrument using the same method mentioned above. 2.4 Buffering Capacity Study Relative buffering capacities of CTS/TPP NPs and CTS/TPP-PAA NPs over a pH range of 10 to 3 pH range were determined using acid−base titration described by Du et al.[27] Initially, the pH value of the 0.33 mg/mL vesicle solutions was adjusted to pH 10 using 0.1 M NaOH. Aliquots of pH 1.5 HCl were then added, and the pH value was measured by a pH meter after each addition. Results and discussion 3.1 Fabrication and characterization of chitosan nanoparticles The procedures of preparing the CTS/TPP-PAA NPs are demonstrated in Figure 1. In the first step, CTS/TPP NPs were prepared by ionic gelation based on the interaction between chitosan and tripolyphosphate.[1,10–12] CTS/TPP NPs were characterized by both DLS and TEM. As shown in Figure 3a and 4a, the size, PDI, and zeta potential of CTS/TPP NPs are about 239.7 nm, 0.056 and 33 mV, respectively. As shown in 3a, the sizes determined by TEM are in the range of 25-45 nm in diameter that are smaller than that obtained by DLS. The discrepancy in the particle diameter is due to the fact that TEM measurements reflect conformations in the dry state while those determined by DLS reflect the hydrodynamic diameter. In the second step, PAA was coated on the surface of CTS/TPP NPs based on electrostatic interaction to render negatively charged surfaces and enhanced stability in biological environments. The electrostatically driven surface modification is controlled by ionic strength, pH value of PAA solution and CTS-to-PAA ratio.[28] The effect of ionic strength on the fabrication of CTS/TPP/PAA NPs was investigated first. CTS/TPP NPs flocculate 4 / 15
once they were redispersed into the PAA solution with as low as 0.05M NaCl. However, CTS/TPP/PAA NPs can be fabricated successfully in PAA solution without NaCl.[29,30] pH 6.0 was found to be the optimal pH for the effective electrostatic interaction between PAA and CTS/TPP NPs and the successful fabrication of CTS/TPP/PAA NPs. Because the global charge densities of chitosan and PAA are affected by the environmental pH and their pKa (the pKa of chitosan and PAA polymer are approximately 6.5 and 4.2, respectively).[31] When the pH of solution went below 4.0, both PAA and chitosan were fully protonated. And, when the solution was neutral or alkaline, PAA was fully ionized and chitosan was unprotonated and hydrophobic. In both of the above situations, the electrostatic interaction between PAA and the amine group on the surface of CTS/TPP NPs is inaccessible. While at pH range of 4.2 to 6.5, both PAA and chitosan were partially ionized/protonated, which enables effective electrostatic interaction between PAA and CTS/TPP NPs. The effect of the ratio of CTS to PAA on the size and zeta potential of CTS/TPP/PAA NPs was investigated. A series of CTS/TPP/PAA NPs with different ratio of CTS to PAA were prepared, and the particle size and zeta potential were measured by DLS, as shown in Figure 2. Increased PAA contents led to more negative zeta potential of CTS/TPP/PAA NPs. A further increase in the CTS:PAA (w:w) ratio led to the disappearance of opalescence in the suspension and a fast flocculation, in which the zeta potential was higher than -20 mV. This is in accord with the reported results of a similar system[32]. The ratio of CTS to PAA was selected to be 1:1 to endow the nanoparticles with enough stability in the subsequent procedures. As shown in Figure 3a, the size and PDI of CTS/TPP/PAA NPs are 230.6 nm and 0.111, respectively.
Figure 2. (a) Effect of ratio of CTS:PAA (w:w) on the mean diameter and zeta potential of CTS/TPP/PAA NPs in MilliQ water. (b) The size and zeta potential of CTS/TPP-PAA NPs in MilliQ water (black) and PBS buffer (pH 7.4, I=150mM) (red) with various CTS:EDC ratios. The PDI range of the nanoparticles with a size below 300 nm is between 0.02 and 0.21. In the third step, the PAA coating was covalently linked onto the surface of chitosan nanoparticles using EDC.[23] The PAA decorated CTS/TPP NPs by covalent bonds is denoted as CTS/TPP-PAA NPs. The effect of the CTS:EDC ratio on the size, zeta potential, and stability of CTS/TPP-PAA nanoparticle was investigated using DLS and TEM. As shown in Figure 2, micrometer-sized aggregates appear when CTS/TPP-PAA NPs with CTS:EDC ratios of 5:1 or 2:1 were re-dispersed in PBS, because of insufficient fixing of PAA polymer on the chitosan nanoparticles. Meanwhile, the size of CTS/TPP-PAA NPs with CTS:EDC ratios of 1:1, 1:2 and 1:5 remains unchanged when the NPs are redispersed in PBS buffer and MilliQ water. In conclusion, the optimal and minimal ratio of CTS:EDC is 1:1. As shown in Figure 3a, the size and PDI of CTS/TPP-PAA NPs are 256.4 nm and 0.140, respectively. As shown in Figure 4a, the zeta potential of CTS/TPP-PAA NPs is about -25 mV. It
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should be mentioned that the zeta potential of CTS/TPP-PAA NPs is lower than that of CTS/TPP/PAA NPs, because of the loss of excess and unbounded PAA polymer on the surface. The yield of ultracentrifugation and redispersion process is calculated by the derived count rate (DCR) measured by DLS. Because photo count rate can used as an indicator of nanoparticle concentration.[33] Therefore, the calculated yields of CTS/TPP, CTS/TPP/PAA, and CTS/TPP-PAA NPs are 89.1%, 84.2% and 83.9%, respectively. It should be mentioned that, in order to enhance the efficiency of coupling between the chitosan nanoparticles and PAA and avoid crosslinking of the chitosan nanoparticles, PAA was electrostatically attached onto the surface of chitosan nanoparticles before the addition of EDC. N-hydroxysuccinimide (NHS) was not used because the NHS-ester is hydrophobic and will lead to the precipitation of nanoparticles.[34]
Figure 3. (a) Hydrodynamic diameter distribution f(Dh) of CTS/TPP, CTS/TPP/PAA and CTS/TPP-PAA NPs in MilliQ Water. TEM images of CTS/TPP NPs (b) and CTS/TPP-PAA NPs (c). In conclusion, there is very slight difference in the size of CTS/TPP, CTS/TPP/PAA and CTS/TPP-PAA NPs with the optimal experimental condition as shown in Figure 2 and Figure 3. This is reasonable because (1) PAA polymer has a low molecular weight, (2) the crosslinking occurred between the PAA layer and the chitosan core. Therefore, the size of CTS/TPP-PAA nanoparticle can be tuned by changing the fabrication conditions of CTS/TPP NPs such as TPP concentration, the CTS-to-TPP ratio, pH of chitosan solution, etc.[11,35–37]. Meanwhile, the zeta potential of CTS/TPP-PAA NPs can be easily tuned by changing the ratio of CTS to PAA. It is worth emphasizing that chitosan-based polyelectrolyte complex (PEC) nanoparticles prepared by electrostatic interactions between chitosan and polyanion have received considerable attention as carrier systems for drug delivery. [1,18,31,38,39] To obtain negative charged chitosan nanoparticles, polyanion should be in excess. However, the negatively charged chitosan PEC nanoparticles can be destabilized in 150 mM salt because of the disruption of the electrostatic interaction, which is dominant in stabilizing the complex.[32] In addition, the polyelectrolyte complex has a large size and a broad size distribution, resulting from the stronger binding of chitosan/polyanion compared with chitosan/TPP and chitosan/pyrophosphate.[3,40] Therefore, surface modification of CTS/TPP NPs with a polyanion is thought to be the optimal approach to obtain chitosan nanoparticles with narrow size distribution, highly compact core and negatively charged surface. 3.2 Stability study of chitosan nanoparticles In drug delivery systems, the stability of nanoparticles is one of the most important factors determining the efficacy of controlled drug delivery.[41] The colloidal stability of the chitosan nanoparticles in MilliQ water,
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PBS and DMEM medium was assessed by DLS. As shown in Figure 4, CTS/TPP, CTS/TPP/PAA and CTS/TPP-PAA NPs are all stable and not aggregate in MilliQ water. And CTS/TPP and CTS/TPP/PAA NPs aggregate in PBS buffer and DMEM medium, while CTS/TPP-PAA NPs remain stable in PBS buffer and DMEM medium, as measured by DLS and optical photography. To further test the suitability of CTS/TPP-PAA NPs for biomedical applications, the colloidal stability of the nanoparticles in PBS buffer at a particle concentration of 1 mg/mL was investigated (Figure 5). Even for a prolonged stability up to 15 days, the hydrodynamic diameter of CTS/TPP-PAA NPs remained unchanged. The excellent stability of the CTS/TPPPAA NPs makes it a promising candidate as drug carrier in biomedical applications.
Figure 4. (a) The size of CTS/TPP, CTS/TPP/PAA and CTS/TPP-PAA NPs in MilliQ water (black) and PBS buffer (red) for 24 h. And the zeta potential of the above nanoparticles in MilliQ water (blue). (b) DLS of CTS/TPP NPs, CTS/TPP/PAA and CTS/TPP-PAA NPs in DMEM medium at 24 h. The inset shows the aggregation of CTS/TPP NPs (left) and the tyndall effect of CTS/TPP-PAA NPs (right) in PBS buffer for 24 h. The PDI range of the nanoparticles with a size below 300 nm is between 0.03 and 0.20. The main cause of the colloidal stability of CTS/TPP-PAA NPs in PBS and simulated body fluid is the balance between electrostatic repulsion and hydrogen-bonding and the stable covalent bonding of PAA to the surface of chitosan nanoparticles.[3,30,36,42,43] Another important reason for the colloidal stability of CTS/TPP-PAA NPs compared with CTS/TPP/PAA NPs is that the hydrogen bonding of the chitosan chains is screened by the PAA polymer.
Figure 5. Size of CTS/TPP-PAA NPs in PBS (pH 7.4, I=150mM). The PDI range of the nanoparticles is between 0.03 and 0.25.
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3.3 Redispersion of lyophilized chitosan nanoparticles Freeze-drying is a good technique to improve the long-term stability of colloidal nanoparticles because water is removed and the chemical instability (hydrolysis of the polymer, drug leakage of nanoparticles and chemical reactivity of the medicine during the storage) can be inhibited.[44] However, the unmodified chitosan nanoparticles tended to aggregate and could not be well re-dispersed after freeze-drying because of the intraand intermolecular hydrogen bonding. [45,46] One method to solve this problem is to modify the chitosan polymer or chitosan nanoparticles by the introduction of polymers such as PEG or groups such as N-acyl groups.[47,48] Another method is to use the freeze-drying protective agent (mannitol, trehalose, polyethyleneglycol, etc) to prevent the unmodified chitosan nanoparticles from aggregating.[49] In our work, the re-dispersion of lyophilized CTS/TPP and CTS/TPP-PAA NPs were characterized by DLS. As shown in Figure 6, CTS/TPP-PAA NPs were easily re-dispersed in water to nano-size, as well as CTS/TPP NPs with 1% mannitol. However, the freeze-dried CTS/TPP NPs without 1% mannitol tended to aggregate and could not be well re-dispersed. In conclusion, similar to the cryoprotectant, PAA polymer on the nanoparticles surface lessens the intra- and intermolecular hydrogen bonding and endows the chitosan nanoparticles with good dispersibility.
Figure 6. Size of CTS/TPP NPs (with and without 1% mannitol) and CTS/TPP-PAA NPs before and after lyophilization. 3.4 Investigation of BSA adsorption For cancer drug delivery, it is desirable to avoid rapid clearance of nanoparticles in the circulatory system. Chemical modification of a nanoparticle surface is one of the most important methods of altering its interaction with biological systems. [50] In our report, the surface charge and hydrophobicity of chitosan nanoparticles was changed by modification with polyanionic PAA, which could endow the nanoparticle with stability in plasma. To assess the stability of chitosan nanoparticles in serum, FITC-BSA was used to investigate the protein adsorption behavior of chitosan nanoparticles. In our work, CTS/TPP or CTS/TPP-PAA NPs were incubated with FITC-BSA for 1.5 h at 37 C, and then separated by centrifugation, and the amount of unbound FITC-BSA in the supernatants was measured by fluorescence spectroscopy (Figure 7). The fluorescence intensity was reduced by 62.4 % after incubation with CTS/TPP NPs. This is reasonable. When exogenous nanoparticles are immersed in biological media, they are rap idly coated by a so-called “protein corona”, which can reduce blood circulation time and hamper cellular
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uptake.[51] In contrast, the fluorescence intensity was increased by 15.3 % after incubation with CTS/TPP-PAA NPs. This experiment and its phenomenon are highly repeatable. To determine whether the fluorescent enhancement was caused by the free polymer (such as CTS and PAA) or the nanoparticles, fluorescent intensity measurement of PAA solutions or chitosan solutions with different concentrations incubated with FITC-BSA undergoing the identical procedure was carried out, but the resultant fluorescent spectrum shows no variation. In the other hand, the fluorescent intensity was increased by 13.9% after incubation with CTS/TPP/PAA NPs. A possible reason of the fluorescent enhancement is that the microenvironment near the fluorescence label (FITCBSA) is changed by the hydrophobic environment of the residual chitosan nanoparticles. Similar result was reported by Sakai[52]. CTS/TPP-PAA NPs presented excellent resistance to protein adsorption because of their hydrophilic and negatively charged surfaces. Therefore, the particle size of CTS/TPP-PAA in DMEM with 10% FBS did not show a significant change compared with in other media (e.g. water or PBS). However, the negative surface charge resulted in decreased cellular uptake of the CTS/TPP-PAA NPs due to the electrostatic repulsion between negatively charged nanoparticles and the negatively charged cell membrane. One method to solve this problem is to reverse the surface charge from negative to positive at the tumor site using stimuli such as pH or temperature that can trigger cell internalization.[13,53–55] An alternative method is to enhance cellular uptake via a ligand-receptor interaction.
Figure 7. Emission spectra of CTS/TPP NPs (blue) and CTS/TPP-PAA NPs (red) after incubation with 167 μg mL-1 FITC-BSA along with emission spectrum of initial FITC-BSA at 167 μg mL-1 (black line). 3.5 Buffering Capacity Study Endosomal escape of the delivered nanoparticles and drugs is essential to improve therapeutic efficiency. Different mechanisms such as pore formation in the endosomal membrane, the pH-buffering effect of protonable groups and fusion into the lipid bilayer of endosomes have been proposed to facilitate the endosomal escape.[56] Polymers with a high buffering capacity, such as PEI[57] , zwitterionic chitosan[4] and PAA[27], could facilitate endosomal escape of the released drugs into the cytosol through the proton sponge mechanism. The relative buffering capacities of CTS/TPP-PAA and CTS/TPP NPs were compared using acid−base titration. As shown in Figure 8, compared with CTS/TPP NPs (the ratio of CTS to PAA is 1:0), CTS/TPP-PAA NPs display much better buffering capacity, which can be attributed to the large number of -COOH groups on the surface of the PAA polymer. CTS/TPP-PAA NPs with different PAA contents were prepared and investigated 9 / 15
for their buffering capacity. As shown in Figure 8, more PAA polymer endows the nanoparticle with better buffering capacity, further confirming the ability of PAA to enhance buffering capacity. Polymers with a high buffering capacity could be protonated in endosomes with low pH, leading to the disruption of the endosome and endosomal escape of drugs.[27] Therefore, the results of the experiment strongly indicate that the PAA coating could facilitate the endosomal escape of drugs and nanoparticles.
Figure 8. Buffering capacity of CTS/TPP-PAA NPs with different ratio of CTS to PAA. It should be mentioned that the pH-sensitivity of the PAA polymer may endow the nanoparticles with both endosomal escape and pH-dependent drug release behavior. Similar results were reported by Wang[58] and Shi[59]. Shi reported polymeric complex micelles with charged channels on the surface, which endow micelles with different drug release rates in different pH environments. A detailed investigation of the pH-dependent release of ionic and non-ionic drugs is now underway. Conclusion In summary, CTS/TPP-PAA NPs with a positively charged core and negatively charged surface were successfully fabricated by surface modification with poly(acrylic acid) on CTS/TPP NPs. PAA endowed CTS/TPP-PAA NPs with excellent stability in plasma and a remarkable buffering capacity, indicating a pontential for rapid endosomal escape. Meanwhile, PAA could be used as a targeting ligand scaffold to enhance cellular uptake. Therefore, the reported chitosan-based nanoparticle could be a promising alternative system for drug delivery.
Acknowledgements This work was supported by the National Natural Science Foundation of China (51433004), PCSIRT (IRT1257), and NFFTBS (No. J1103306). We thank Prof. Deling Kong and Prof Jun Yang, Nankai University, for their help with the whole cell experiments.
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CTS/TPP-PAA nanoparticles with a positively charged core and negatively charged surface were successfully fabricated. And PAA endowed CTS/TPP-PAA NPs with excellent stability in plasma and rapid endosomal escape.
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S0939-6411(16)30845-1 http://dx.doi.org/10.1016/j.ejpb.2016.11.020 EJPB 12355
To appear in:
European Journal of Pharmaceutics and Biopharmaceutics
Received Date: Revised Date: Accepted Date:
12 June 2016 17 November 2016 20 November 2016
Please cite this article as: Y. Wu, J. Wu, J. Cao, Y. Zhang, Z. Xu, X. Qin, W. Wang, Z. Yuan, Facile fabrication of poly(acrylic acid) coated chitosan nanoparticles with improved stability in biological environments, European Journal of Pharmaceutics and Biopharmaceutics (2016), doi: http://dx.doi.org/10.1016/j.ejpb.2016.11.020
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Facile fabrication of poly(acrylic acid) coated chitosan nanoparticles with improved stability in biological environments Yukun Wu,† Jing Wu,† Jing Cao,† Yajie Zhang,† Zhe Xu,† Xiuyi Qin,† Wei Wang,† Zhi Yuan*,†,‡ †
Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, and
‡
Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China * Corresponding author E-mail address: [email protected] Tel.: +86-22-23501164 Fax: +86-22-23503510 Abstract Chitosan is one of the most important and commonly used natural polysaccharides in drug delivery for its biocompatible and biodegradable properties. However, poor blood circulation of the chitosan nanoparticles due to their cationic nature is one of the major bottlenecks of chitosan-based drug delivery systems. To address this problem, a versatile platform based on poly(acrylic acid) (PAA) coated ionically cross-linked chitosan/tripolyphosphate nanoparticles (CTS/TPP-PAA NPs), is reported. The zeta potentials of CTS/TPP and CTS/TPP-PAA NPs are approximately 33 mV and -25 mV, respectively. CTS/TPP NPs quickly aggregate in PBS (phosphate buffered saline) and DMEM (Dulbecco's modified Eagle's medium). Conversely, CTS/TPPPAA NPs exhibit excellent colloidal stability in plasma solution for more than 24 h. The PAA coating also endows CTS/TPP-PAA NPs with decreased protein adsorption capacity and improved buffering capacity. More importantly, the residual carboxyl and amino groups on CTS/TPP-PAA NPs provide abundant reactive sites for further functional modifications. Therefore, the CTS/TPP-PAA NPs reported here may be useful as an alternative drug delivery system.
Keywords Chitosan, poly(acrylic acid), surface modification, plasma stability, endosomal escape, drug delivery Introduction Chitosan has been widely utilized for a variety of medical applications, such as cell imaging and drug delivery systems for peptides, genes and low-molecular-weight drugs.[1] As the only known naturally occurring polycationic polysaccharide, chitosan has versatile characteristics, such as bioavailability, biocompatibility, biodegradability, low immunogenicity, and high charge density.[2] Chitosan nanoparticles can be prepared by chitosan-drug conjugates, ionically or covalently cross-linked chitosan, or amphiphilic chitosan derivatives.[3– 10] Among them, ionic cross-linking of chitosan using tripolyphosphate (TPP) as the crosslinker has numerous advantages, such as mild, convenient, non-toxic and organic solvent free preparation conditions.[3,11,12] Despite their biocompatibility and biodegradability, chitosan nanoparticles, like most of the positively charged nanoparticles, always interact strongly with serum components, which causes severe aggregation and rapid clearance from circulation and limits their in vivo application.[13,14] Therefore, tailoring the chitosan nanoparticles towards greater stability in biological environments is important for tumor targeting and
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intracellular drug delivery. The most frequently used method to overcome this drawback is hydrophilic modification of chitosan, using polymers and molecules such as β-cyclodextrin,[15] PEG,[7] and succinic anhydride,[4,6] which enhance chitosan’s solubility in slightly acid and neutral media and confer stability to the chitosan nanoparticles. However, hydrophilic modification of the chitosan backbone can compromise the hydrophobic and cationic integrity of the chitosan core, which can lead to a reduced hydrophobic drug loading capacity[16,17]. At the same time, methods of stabilizing chitosan nanoparticles against aggregation through surface decoration with hydrophilic polymers or macromolecules have rarely been reported.[18] In the present work, we aimed to develop a facile and versatile approach towards the formation of chitosan nanoparticles that were stable in biological media. (Figure 1) First, TPP cross-linked chitosan (CTS/TPP) nanoparticles were fabricated by the standard ionic gelation technique. Second, polyanion poly(acrylic acid) (PAA) was attached to the surface of chitosan nanoparticles through an electrostatic interaction between positively charged protonated chitosan chains and the negatively charged PAA polymer. Finally, the PAA polymer was fixed onto the surface of the CTS/TPP core using water-soluble carbodiimide (EDC) at room temperature. This chitosan-based nanoparticle has some interesting features: (1) the residual carboxyl and amino groups on PAA-CTS/TPP NPs provide abundant reactive sites for further functional modifications. (2) The post-modification of CTS/TPP NPs did not impair the integrity of the chitosan core, which is beneficial for increasing the drug loading capacity and gene condensing capacity. (3) The CTS/TPP-PAA NPs exhibited excellent colloidal stability in PBS and plasma solution. (4) The electrostatic modification of CTS/TPP nanoparticles is simple and versatile, and could easily be applied to a variety of chitosan nanoparticles and analogous nanoparticles.
Figure 1. Scheme for preparation procedure of chitosan-based nanoparticles. . Experimental 2.1 Materials Chitosan was purchased from TCI Co. Ltd. (Japan), with deacetylation degrees of 75.0 to 85.0 %. FITC-labeled chitosan (FITC-CTS) was synthesized according to our previous studies.[10] Poly(acrylic acid) (PAA, molecular weight of 1800 Da), folate (folic acid), sodium tripolyphosphate (TPP), fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) and 1-dimethylaminopropyl-ethylcarbodiimide hydrochloride (EDC) were all purchased from Aladdin (Shanghai, China) and used as received. The other chemical reagents used in this study were all commercially acquired from Tianjin Chemical Reagent Company (Tianjin, China) with analytical reagent grade.
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The human liver carcinoma cell line (HepG2) was purchased from the Shanghai Institute of Biochemistry and Cell Biology cell bank. DMEM medium, FBS, trypsin and other biological reagents were all purchased from DingGuo Biotech. Co. (Shanghai, China). 2.2 Fabrication of chitosan nanoparticles Fabrication of PAA coated chitosan nanoparticles were achieved in three steps, as illustrated in Figure 1. TPP cross-linked chitosan nanoparticles (CTS/TPP NPs) were prepared based on the ionic gelation interaction between positively charged chitosan and negatively charged TPP according to a previously reported method.[19–22] Briefly, chitosan was dissolved overnight in aqueous acetic acid (1 wt %) at a concentration of 1 mg/mL, and the pH was adjusted to 6.0 using sodium hydroxide solution. Under intensive stirring, TPP solution (1 mg/ml) was added dropwise (1 drop per 1-2 s) to the CTS solution in an ice water bath. The solution was stirred for 24 h at room temperature before use. Nanoparticles were ultra-centrifuged at 15,000 rpm at 4 oC for 1.5 h in an ultracentrifuge (Sigma 3K30, UK) and redispersed using a probe ultrasonic processor (Scientz-IID, Ningbo Scientz Biotech., Ningbo, China) with 10W output power, 25KHz ultrasonic frequency for 5 min. Poly(acrylic acid) coated chitosan nanoparticles (CTS/TPP/PAA NPs) were prepared based on an electrostatic interaction between chitosan and poly(acrylic acid), according to the layer-by-layer (LBL) method.[23] Briefly, CTS/TPP NPs were ultra-centrifuged at 15,000 rpm at 4 oC for 1.5 h in an ultracentrifuge (Sigma 3K30, UK) and redispersed in an aqueous solution of 10mg/mL PAA (molecular weight 1800 Da, pH=6.0) using the same procedure mentioned above, during which the PAA was electrostatically attached to the CTS/TPP NPs. The nanoparticles were then separated from the PAA solution and redispersed in MilliQ water (Millipore Milli-Q purification system, MA, USA) using the same procedure mentioned above. Poly(acrylic acid) conjugated chitosan nanoparticles (CTS/TPP-PAA NPs) were prepared by conjugation between the PAA coating and CTS/TPP core using a water-soluble carbodiimide (EDC) as the cross-linker, according to a previously described method.[24,25] Briefly, an EDC aqueous solution (10 mg/mL) was mixed with the CTS/TPP/PAA NPs (1 mg/mL) with sonication and reacted at room temperature overnight to fix the PAA coating via the amide bond between the carboxyl group of PAA and the amino group on the chitosan nanoparticles surface. Subsequently, nanoparticles were collected by centrifugation at 15,000 rpm for 1.5 h. The supernatants were discarded and the pellet was redispersed in MilliQ water. 2.3 Evaluation of nanoparticles stability 2.3.1 Size measurement of nanoparticles The size, zeta potential of nanoparticles were determined using a Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK) equipped with 4 mW He Ne laser at a scattering angle of 90o at 532 nm at 25 C. Polydispersity index (PDI) was also measured to determine particle size distribution. The typical nanoparticle concentration used was 0.1mg/mL in MilliQ water or PBS. The solutions were filtered using a 0.45 micron filter prior to analysis. All the measurements were repeated three times, and an average value was reported. The stability of nanoparticles under simulated physiological conditions was monitored by size measurements at different time points. Chitosan nanoparticles were ultra-centrifuged at 15,000 rpm for 1.5 h, and the obtained pellets were redispersed in MilliQ water, PBS (pH 7.4, 10mM, I=150 mM) and DMEM cell culture medium with 10% FBS, respectively. The nanoparticles were incubated at 37 C under agitation (100 rpm).
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The size and morphology of nanoparticles were measured by transmission electron microscopy (TEM) on a Tecnai G2 F20 S-Twin electron microscopy center (FEI Company, America). To prepare the TEM samples, the sample solution was dropped onto a carbon-coated copper grid, stained with 2% (w/v) phosphotungstic acid aqueous solution, and dried slowly in air. The acceleration voltage was 100 kV. 2.3.2 BSA absorption assay The interaction between bovine serum albumin (BSA) and the chitosan nanoparticles was investigated according to the literature procedure.[26] Approximately 0.4 mL of a dispersion of CTS/TPP and CTS/TPP-PAA nanoparticles (0.5 mg/mL) was suspended in 0.2 mL of aqueous solution with FITC-BSA at an initial concentration of 0.05 mg/mL. The samples were then shaken for 1.5 h at 37 C. Nanoparticles were separated from unbound FITC-BSA by ultracentrifugation at 15,000 rpm and 4 C for 1.5 h. The fluorescence intensity of the supernatant was measured using a fluorescence spectrometer (F-7000, Hitachi, Japan). A solution of 167 μg mL-1 FITC-BSA in MilliQ water was utilized as a control. Samples were excited at a fixed wavelength of 488 nm and spectra were recorded in a wavelength range between 500 and 550 nm. The input/output slit was 5 nm. The PMT voltage was 700V. 2.3.3 Effect of freeze-drying on the stability of chitosan nanoparticles CTS/TPP and CTS/TPP-PAA NPs (1 mg/mL) with or without the commercial cryoprotectant mannitol (1%) were freeze-dried using a Scientz-10N lyophilizer (Xin Zhi company, China). Solutions were firstly frozen to 20 °C for 5 hours. Frozen solutions were then desiccated under vacuum (-0.1MPa) with a primary drying stage at -20°C for 12 hours, and a secondary drying stage at room temperature for 24 hours. The freeze-dried samples were finally redispersed in MilliQ water using a probe ultrasonic processor (Scientz-IID, Ningbo Scientz Biotech., Ningbo, China). And the nanoparticle size and size distribution were measured by the DLS instrument using the same method mentioned above. 2.4 Buffering Capacity Study Relative buffering capacities of CTS/TPP NPs and CTS/TPP-PAA NPs over a pH range of 10 to 3 pH range were determined using acid−base titration described by Du et al.[27] Initially, the pH value of the 0.33 mg/mL vesicle solutions was adjusted to pH 10 using 0.1 M NaOH. Aliquots of pH 1.5 HCl were then added, and the pH value was measured by a pH meter after each addition. Results and discussion 3.1 Fabrication and characterization of chitosan nanoparticles The procedures of preparing the CTS/TPP-PAA NPs are demonstrated in Figure 1. In the first step, CTS/TPP NPs were prepared by ionic gelation based on the interaction between chitosan and tripolyphosphate.[1,10–12] CTS/TPP NPs were characterized by both DLS and TEM. As shown in Figure 3a and 4a, the size, PDI, and zeta potential of CTS/TPP NPs are about 239.7 nm, 0.056 and 33 mV, respectively. As shown in 3a, the sizes determined by TEM are in the range of 25-45 nm in diameter that are smaller than that obtained by DLS. The discrepancy in the particle diameter is due to the fact that TEM measurements reflect conformations in the dry state while those determined by DLS reflect the hydrodynamic diameter. In the second step, PAA was coated on the surface of CTS/TPP NPs based on electrostatic interaction to render negatively charged surfaces and enhanced stability in biological environments. The electrostatically driven surface modification is controlled by ionic strength, pH value of PAA solution and CTS-to-PAA ratio.[28] The effect of ionic strength on the fabrication of CTS/TPP/PAA NPs was investigated first. CTS/TPP NPs flocculate 4 / 15
once they were redispersed into the PAA solution with as low as 0.05M NaCl. However, CTS/TPP/PAA NPs can be fabricated successfully in PAA solution without NaCl.[29,30] pH 6.0 was found to be the optimal pH for the effective electrostatic interaction between PAA and CTS/TPP NPs and the successful fabrication of CTS/TPP/PAA NPs. Because the global charge densities of chitosan and PAA are affected by the environmental pH and their pKa (the pKa of chitosan and PAA polymer are approximately 6.5 and 4.2, respectively).[31] When the pH of solution went below 4.0, both PAA and chitosan were fully protonated. And, when the solution was neutral or alkaline, PAA was fully ionized and chitosan was unprotonated and hydrophobic. In both of the above situations, the electrostatic interaction between PAA and the amine group on the surface of CTS/TPP NPs is inaccessible. While at pH range of 4.2 to 6.5, both PAA and chitosan were partially ionized/protonated, which enables effective electrostatic interaction between PAA and CTS/TPP NPs. The effect of the ratio of CTS to PAA on the size and zeta potential of CTS/TPP/PAA NPs was investigated. A series of CTS/TPP/PAA NPs with different ratio of CTS to PAA were prepared, and the particle size and zeta potential were measured by DLS, as shown in Figure 2. Increased PAA contents led to more negative zeta potential of CTS/TPP/PAA NPs. A further increase in the CTS:PAA (w:w) ratio led to the disappearance of opalescence in the suspension and a fast flocculation, in which the zeta potential was higher than -20 mV. This is in accord with the reported results of a similar system[32]. The ratio of CTS to PAA was selected to be 1:1 to endow the nanoparticles with enough stability in the subsequent procedures. As shown in Figure 3a, the size and PDI of CTS/TPP/PAA NPs are 230.6 nm and 0.111, respectively.
Figure 2. (a) Effect of ratio of CTS:PAA (w:w) on the mean diameter and zeta potential of CTS/TPP/PAA NPs in MilliQ water. (b) The size and zeta potential of CTS/TPP-PAA NPs in MilliQ water (black) and PBS buffer (pH 7.4, I=150mM) (red) with various CTS:EDC ratios. The PDI range of the nanoparticles with a size below 300 nm is between 0.02 and 0.21. In the third step, the PAA coating was covalently linked onto the surface of chitosan nanoparticles using EDC.[23] The PAA decorated CTS/TPP NPs by covalent bonds is denoted as CTS/TPP-PAA NPs. The effect of the CTS:EDC ratio on the size, zeta potential, and stability of CTS/TPP-PAA nanoparticle was investigated using DLS and TEM. As shown in Figure 2, micrometer-sized aggregates appear when CTS/TPP-PAA NPs with CTS:EDC ratios of 5:1 or 2:1 were re-dispersed in PBS, because of insufficient fixing of PAA polymer on the chitosan nanoparticles. Meanwhile, the size of CTS/TPP-PAA NPs with CTS:EDC ratios of 1:1, 1:2 and 1:5 remains unchanged when the NPs are redispersed in PBS buffer and MilliQ water. In conclusion, the optimal and minimal ratio of CTS:EDC is 1:1. As shown in Figure 3a, the size and PDI of CTS/TPP-PAA NPs are 256.4 nm and 0.140, respectively. As shown in Figure 4a, the zeta potential of CTS/TPP-PAA NPs is about -25 mV. It
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should be mentioned that the zeta potential of CTS/TPP-PAA NPs is lower than that of CTS/TPP/PAA NPs, because of the loss of excess and unbounded PAA polymer on the surface. The yield of ultracentrifugation and redispersion process is calculated by the derived count rate (DCR) measured by DLS. Because photo count rate can used as an indicator of nanoparticle concentration.[33] Therefore, the calculated yields of CTS/TPP, CTS/TPP/PAA, and CTS/TPP-PAA NPs are 89.1%, 84.2% and 83.9%, respectively. It should be mentioned that, in order to enhance the efficiency of coupling between the chitosan nanoparticles and PAA and avoid crosslinking of the chitosan nanoparticles, PAA was electrostatically attached onto the surface of chitosan nanoparticles before the addition of EDC. N-hydroxysuccinimide (NHS) was not used because the NHS-ester is hydrophobic and will lead to the precipitation of nanoparticles.[34]
Figure 3. (a) Hydrodynamic diameter distribution f(Dh) of CTS/TPP, CTS/TPP/PAA and CTS/TPP-PAA NPs in MilliQ Water. TEM images of CTS/TPP NPs (b) and CTS/TPP-PAA NPs (c). In conclusion, there is very slight difference in the size of CTS/TPP, CTS/TPP/PAA and CTS/TPP-PAA NPs with the optimal experimental condition as shown in Figure 2 and Figure 3. This is reasonable because (1) PAA polymer has a low molecular weight, (2) the crosslinking occurred between the PAA layer and the chitosan core. Therefore, the size of CTS/TPP-PAA nanoparticle can be tuned by changing the fabrication conditions of CTS/TPP NPs such as TPP concentration, the CTS-to-TPP ratio, pH of chitosan solution, etc.[11,35–37]. Meanwhile, the zeta potential of CTS/TPP-PAA NPs can be easily tuned by changing the ratio of CTS to PAA. It is worth emphasizing that chitosan-based polyelectrolyte complex (PEC) nanoparticles prepared by electrostatic interactions between chitosan and polyanion have received considerable attention as carrier systems for drug delivery. [1,18,31,38,39] To obtain negative charged chitosan nanoparticles, polyanion should be in excess. However, the negatively charged chitosan PEC nanoparticles can be destabilized in 150 mM salt because of the disruption of the electrostatic interaction, which is dominant in stabilizing the complex.[32] In addition, the polyelectrolyte complex has a large size and a broad size distribution, resulting from the stronger binding of chitosan/polyanion compared with chitosan/TPP and chitosan/pyrophosphate.[3,40] Therefore, surface modification of CTS/TPP NPs with a polyanion is thought to be the optimal approach to obtain chitosan nanoparticles with narrow size distribution, highly compact core and negatively charged surface. 3.2 Stability study of chitosan nanoparticles In drug delivery systems, the stability of nanoparticles is one of the most important factors determining the efficacy of controlled drug delivery.[41] The colloidal stability of the chitosan nanoparticles in MilliQ water,
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PBS and DMEM medium was assessed by DLS. As shown in Figure 4, CTS/TPP, CTS/TPP/PAA and CTS/TPP-PAA NPs are all stable and not aggregate in MilliQ water. And CTS/TPP and CTS/TPP/PAA NPs aggregate in PBS buffer and DMEM medium, while CTS/TPP-PAA NPs remain stable in PBS buffer and DMEM medium, as measured by DLS and optical photography. To further test the suitability of CTS/TPP-PAA NPs for biomedical applications, the colloidal stability of the nanoparticles in PBS buffer at a particle concentration of 1 mg/mL was investigated (Figure 5). Even for a prolonged stability up to 15 days, the hydrodynamic diameter of CTS/TPP-PAA NPs remained unchanged. The excellent stability of the CTS/TPPPAA NPs makes it a promising candidate as drug carrier in biomedical applications.
Figure 4. (a) The size of CTS/TPP, CTS/TPP/PAA and CTS/TPP-PAA NPs in MilliQ water (black) and PBS buffer (red) for 24 h. And the zeta potential of the above nanoparticles in MilliQ water (blue). (b) DLS of CTS/TPP NPs, CTS/TPP/PAA and CTS/TPP-PAA NPs in DMEM medium at 24 h. The inset shows the aggregation of CTS/TPP NPs (left) and the tyndall effect of CTS/TPP-PAA NPs (right) in PBS buffer for 24 h. The PDI range of the nanoparticles with a size below 300 nm is between 0.03 and 0.20. The main cause of the colloidal stability of CTS/TPP-PAA NPs in PBS and simulated body fluid is the balance between electrostatic repulsion and hydrogen-bonding and the stable covalent bonding of PAA to the surface of chitosan nanoparticles.[3,30,36,42,43] Another important reason for the colloidal stability of CTS/TPP-PAA NPs compared with CTS/TPP/PAA NPs is that the hydrogen bonding of the chitosan chains is screened by the PAA polymer.
Figure 5. Size of CTS/TPP-PAA NPs in PBS (pH 7.4, I=150mM). The PDI range of the nanoparticles is between 0.03 and 0.25.
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3.3 Redispersion of lyophilized chitosan nanoparticles Freeze-drying is a good technique to improve the long-term stability of colloidal nanoparticles because water is removed and the chemical instability (hydrolysis of the polymer, drug leakage of nanoparticles and chemical reactivity of the medicine during the storage) can be inhibited.[44] However, the unmodified chitosan nanoparticles tended to aggregate and could not be well re-dispersed after freeze-drying because of the intraand intermolecular hydrogen bonding. [45,46] One method to solve this problem is to modify the chitosan polymer or chitosan nanoparticles by the introduction of polymers such as PEG or groups such as N-acyl groups.[47,48] Another method is to use the freeze-drying protective agent (mannitol, trehalose, polyethyleneglycol, etc) to prevent the unmodified chitosan nanoparticles from aggregating.[49] In our work, the re-dispersion of lyophilized CTS/TPP and CTS/TPP-PAA NPs were characterized by DLS. As shown in Figure 6, CTS/TPP-PAA NPs were easily re-dispersed in water to nano-size, as well as CTS/TPP NPs with 1% mannitol. However, the freeze-dried CTS/TPP NPs without 1% mannitol tended to aggregate and could not be well re-dispersed. In conclusion, similar to the cryoprotectant, PAA polymer on the nanoparticles surface lessens the intra- and intermolecular hydrogen bonding and endows the chitosan nanoparticles with good dispersibility.
Figure 6. Size of CTS/TPP NPs (with and without 1% mannitol) and CTS/TPP-PAA NPs before and after lyophilization. 3.4 Investigation of BSA adsorption For cancer drug delivery, it is desirable to avoid rapid clearance of nanoparticles in the circulatory system. Chemical modification of a nanoparticle surface is one of the most important methods of altering its interaction with biological systems. [50] In our report, the surface charge and hydrophobicity of chitosan nanoparticles was changed by modification with polyanionic PAA, which could endow the nanoparticle with stability in plasma. To assess the stability of chitosan nanoparticles in serum, FITC-BSA was used to investigate the protein adsorption behavior of chitosan nanoparticles. In our work, CTS/TPP or CTS/TPP-PAA NPs were incubated with FITC-BSA for 1.5 h at 37 C, and then separated by centrifugation, and the amount of unbound FITC-BSA in the supernatants was measured by fluorescence spectroscopy (Figure 7). The fluorescence intensity was reduced by 62.4 % after incubation with CTS/TPP NPs. This is reasonable. When exogenous nanoparticles are immersed in biological media, they are rap idly coated by a so-called “protein corona”, which can reduce blood circulation time and hamper cellular
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uptake.[51] In contrast, the fluorescence intensity was increased by 15.3 % after incubation with CTS/TPP-PAA NPs. This experiment and its phenomenon are highly repeatable. To determine whether the fluorescent enhancement was caused by the free polymer (such as CTS and PAA) or the nanoparticles, fluorescent intensity measurement of PAA solutions or chitosan solutions with different concentrations incubated with FITC-BSA undergoing the identical procedure was carried out, but the resultant fluorescent spectrum shows no variation. In the other hand, the fluorescent intensity was increased by 13.9% after incubation with CTS/TPP/PAA NPs. A possible reason of the fluorescent enhancement is that the microenvironment near the fluorescence label (FITCBSA) is changed by the hydrophobic environment of the residual chitosan nanoparticles. Similar result was reported by Sakai[52]. CTS/TPP-PAA NPs presented excellent resistance to protein adsorption because of their hydrophilic and negatively charged surfaces. Therefore, the particle size of CTS/TPP-PAA in DMEM with 10% FBS did not show a significant change compared with in other media (e.g. water or PBS). However, the negative surface charge resulted in decreased cellular uptake of the CTS/TPP-PAA NPs due to the electrostatic repulsion between negatively charged nanoparticles and the negatively charged cell membrane. One method to solve this problem is to reverse the surface charge from negative to positive at the tumor site using stimuli such as pH or temperature that can trigger cell internalization.[13,53–55] An alternative method is to enhance cellular uptake via a ligand-receptor interaction.
Figure 7. Emission spectra of CTS/TPP NPs (blue) and CTS/TPP-PAA NPs (red) after incubation with 167 μg mL-1 FITC-BSA along with emission spectrum of initial FITC-BSA at 167 μg mL-1 (black line). 3.5 Buffering Capacity Study Endosomal escape of the delivered nanoparticles and drugs is essential to improve therapeutic efficiency. Different mechanisms such as pore formation in the endosomal membrane, the pH-buffering effect of protonable groups and fusion into the lipid bilayer of endosomes have been proposed to facilitate the endosomal escape.[56] Polymers with a high buffering capacity, such as PEI[57] , zwitterionic chitosan[4] and PAA[27], could facilitate endosomal escape of the released drugs into the cytosol through the proton sponge mechanism. The relative buffering capacities of CTS/TPP-PAA and CTS/TPP NPs were compared using acid−base titration. As shown in Figure 8, compared with CTS/TPP NPs (the ratio of CTS to PAA is 1:0), CTS/TPP-PAA NPs display much better buffering capacity, which can be attributed to the large number of -COOH groups on the surface of the PAA polymer. CTS/TPP-PAA NPs with different PAA contents were prepared and investigated 9 / 15
for their buffering capacity. As shown in Figure 8, more PAA polymer endows the nanoparticle with better buffering capacity, further confirming the ability of PAA to enhance buffering capacity. Polymers with a high buffering capacity could be protonated in endosomes with low pH, leading to the disruption of the endosome and endosomal escape of drugs.[27] Therefore, the results of the experiment strongly indicate that the PAA coating could facilitate the endosomal escape of drugs and nanoparticles.
Figure 8. Buffering capacity of CTS/TPP-PAA NPs with different ratio of CTS to PAA. It should be mentioned that the pH-sensitivity of the PAA polymer may endow the nanoparticles with both endosomal escape and pH-dependent drug release behavior. Similar results were reported by Wang[58] and Shi[59]. Shi reported polymeric complex micelles with charged channels on the surface, which endow micelles with different drug release rates in different pH environments. A detailed investigation of the pH-dependent release of ionic and non-ionic drugs is now underway. Conclusion In summary, CTS/TPP-PAA NPs with a positively charged core and negatively charged surface were successfully fabricated by surface modification with poly(acrylic acid) on CTS/TPP NPs. PAA endowed CTS/TPP-PAA NPs with excellent stability in plasma and a remarkable buffering capacity, indicating a pontential for rapid endosomal escape. Meanwhile, PAA could be used as a targeting ligand scaffold to enhance cellular uptake. Therefore, the reported chitosan-based nanoparticle could be a promising alternative system for drug delivery.
Acknowledgements This work was supported by the National Natural Science Foundation of China (51433004), PCSIRT (IRT1257), and NFFTBS (No. J1103306). We thank Prof. Deling Kong and Prof Jun Yang, Nankai University, for their help with the whole cell experiments.
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CTS/TPP-PAA nanoparticles with a positively charged core and negatively charged surface were successfully fabricated. And PAA endowed CTS/TPP-PAA NPs with excellent stability in plasma and rapid endosomal escape.
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