Article pubs.acs.org/Langmuir
Development of Zwitterionic Polymer-Based Doxorubicin Conjugates: Tuning the Surface Charge To Prolong the Circulation and Reduce Toxicity Zhen Wang,† Guanglong Ma,† Juan Zhang,† Weifeng Lin,† Fangqin Ji,† Matthew T. Bernards,‡ and Shengfu Chen*,† †
State Key Laboratory of Chemical Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, P. R. China ‡ Department of Chemical Engineering, University of Missouri, Columbia, Missouri 65211, United States ABSTRACT: Polymer−drug conjugates are commonly used as nano drug vehicles (NDVs) to delivery anticancer drugs. Zwitterionic polymers are ideal candidates to conjugate drugs because they show higher resistance to nonspecific protein adsorption in complex media than that of nonionic watersoluble polymers, such as poly(ethylene glycol). However, the charge balance characteristics of zwitterionic polymers used as NDVs will be broken from the inclusion of additional charged groups brought by conjugated drugs or functional groups, leading to the loss of resistance to protein adsorption. Consequently, the nonspecific protein adsorption on drug carriers will cause fast clearance from the blood system, an immune response, or even severe systemic toxicity. To overcome this drawback, a model zwitterionic polymer, poly(carboxybetaine methacrylate) (pCBMA), was modified by the introduction of a negatively charged component, to neutralize the positive charge provided by the model drug, doxorubicin (DOX). A DOX-conjugated NDV which possesses excellent resistance to nonspecific protein adsorption was achieved by the formation of a strongly hydrated pCBMA shell with a slightly negative surface charge. This kind of DOX-conjugated NDV exhibited reduced cytotoxicity and prolonged circulation time, and it accelerated DOX release under mild acid conditions. In tumor-bearing mouse studies a 55% tumor-inhibition rate was achieved without causing any body weight loss. These results indicate the importance of charge tuning in zwitterionic polymer-based NDVs.
1. INTRODUCTION
After years of research on nonfouling materials, it is recognized that the capability of the water-soluble polymers used for NDVs to reduce nonspecific protein adsorption could be the reason for the improved drug efficacy. However, zwitterionic materials have shown even better resistance to nonspecific protein adsorption than the above water-soluble polymers, suggesting that zwitterionic materials are a promising alternative for NDVs. The low net charge density, homogeneous charge distribution, and strong surface hydration are considered to be the intrinsic features of zwitterionic materials that result in the resistance to nonspecific protein adsorption.23 R e c e n t ly , z w i t t e r i o n i c m a t e r i a l s , s u c h a s p o l y (methacryloyloxyethyl phosphorylcholine) (pMPC), poly(carboxybetaine) (pCB), and poly(sulfobetaine) (pSB), have been developed and applied to a broad range of biomedical and engineering materials.24−27 In particular, pCB shows great potential in drug delivery systems, due to its unique properties:
Most anticancer drugs are low molecular weight compounds with poor water solubility, rapid blood clearance, low tumor selectivity, and severe side effects.1 To overcome these defects, nano drug vehicles (NDVs) conjugated or encapsulated with drug and modified with water-soluble polymer carriers such as poly(amino acids), 2−4 poly(vinylpyrrolidone), 5−7 poly(ethylene glycol) (PEG), 8 −1 3 N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers14−16 and polysaccharides17−19 were developed. These systems have shown enhanced water solubility and stability, prolonged blood circulation, and improved tumor tissue targeting by either passive accumulation via the enhanced permeability and retention (EPR) effect20 or active targeting using targeting ligands (sugars, peptides, and antibodies).21 However, the long-term stability of nanoparticles in complex media is crucial for achieving long circulation times, and it is one of the most complex problems, as it is difficult to control defects.22 Furthermore, NDV stability is primarily determined by their surface coating composition and surface charge. © 2014 American Chemical Society
Received: January 8, 2014 Revised: March 6, 2014 Published: March 11, 2014 3764
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Scheme 1. Molecular Structure of the Copolymer−DOX NDV and a Representative Illustration of the NDVs with Different Net Charges That Were Formed in This Study
toxicity for this NDV were expected and demonstrated in this investigation.
it is extremely hydrophilic, it is ultraresistant to nonspecific protein adsorption (150 nm intensity peak in DLS was observed after all the aggregates precipitated.
visible aggregates could be observed in the cuvettes after incubation in the Fg solution and FBS. These results indicate that copolymer 3−DOX micelles have the best stability in complex biologically relevant media and should have higher biocompatibility in vivo as compared to the other two micelle formulations. The excellent stability of the copolymer 3−DOX micelles is attributed to the protein-resistant pCBMA surface and the slightly negatively charged surface of the polymeric micelles. In the copolymer 1−DOX micelles, the extra positive charges on 3769
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micelles is most likely caused by the surface charge and limited cellular interactions. All the polymer precursors showed very low in vitro cytotoxicity, the cell viabilities of COS-7 cells incubated with polymer precursor (300 μg/mL) for 24 h were higher than 95% (data not given). The in vitro cytotoxicities of free DOX and the polymer−DOX micelles were determined with COS-7 cells using an MTT assay. As shown in Figure 5, the copolymer 1− DOX micelles exhibit cytotoxicity greater than that of the free DOX alone. This result is quite different with DOX-conjugated NDVs based on poly(HPMA)37 and PEG polymer systems.30 The cytotoxicity of those micelles were found to be lower than the free drug due to the slower internalization rate of the micelles and the time-consuming process to release the DOX from the NDVs. The higher cytotoxicity of the micelles of copolymer 1−DOX conjugates is likely caused by the positive charge of the micelle. It is known that positively charged micelles can accelerate cell uptake and directly damage cell membranes through attractive interaction with the negatively charged cell membrane.39−41 The polymer−DOX micelles containing SMA showed much lower cytotoxicity. These results demonstrate that pCBMA-based DOX NDVs require an additional negative component to neutralize the overall NDV charge to prevent negative impacts on cells throughout the body other than the targeted cancer cells. This also suggests that other charge-balanced zwitterionic polymer-based carriers will face the same challenges after they are conjugated with positively charged drugs or functional groups. 3.6. Blood Clearance of the Polymer−DOX Micelles. The blood clearance of the DOX and polymer−DOX micelles was monitored in female ICR mice. The mice were iv injected with DOX or the polymer−DOX micelles with a dosage of 10 mg/kg DOX or DOX-equivalent, and the DOX concentration in the blood was tracked to further evaluate the in vivo stability and safety of the micelles. The results are shown in Figure 6A. In agreement with similar studies,42 DOX was rapidly cleared from the blood due to its small molecular size and hydrophobicity. Severe in vivo acute toxicity was seen with the copolymer 1−DOX micelles, as all the mice died within 2 h of the iv injection. This result agrees with the in vitro stability of the NDVs in complex biological media and the in vitro cytotoxicity results. The likely cause of death was fatal blood clotting induced by the positively charged copolymer 1−DOX micelles based on the clear Fg aggregation that was observed during the in vitro experiment (Figure 2C). The copolymer 2− DOX micelles showed a slight increase in their circulation time. Lastly, the circulation time of the negatively charged copolymer 3−DOX micelles was greatly prolonged as compared to the free DOX. These results also agree with the stability studies (Figure 2B and Figure 2C), the MCF-7 internalization studies, and the COS-7 cytotoxicity studies. In short, the incorporation of a negatively charged component to neutralize the positive charge induced by the encapsulated drug is a feasible method to improve the biocompatibility of zwitterionic polymer-based drug delivery vehicles. Moreover, the stability of NDVs in FBS and Fg solutions is a useful screening tool, although it is does not guarantee long circulation times for the NDV in the bloodstream. 3.7. In Vivo Antitumor Study. The in vivo antitumor activity of the polymer−DOX micelles with a xenograft tumor model in female BALB/c nude mice and the results are shown in Figure 6B and 6C. Polymer−DOX micelles or free DOX were iv injected at a DOX or DOX-equivalent dosage of 4 mg/
Figure 3. Mean ± SD (N = 3) of the in vitro release of DOX from copolymer 1−DOX micelles (A), copolymer 2−DOX micelles (B), and copolymer 3−DOX micelles (C) in pH 7.4 and pH 5.0 buffer solution at 37 °C.
formulations. It is believed that the negative surface charge on the copolymer 3−DOX micelles is the key reason for this low uptake amount, possibly due to a reduction in the occurrence of interactions with the MCF-7 cells. At the same time, the size and shape of the micelle drug carrier38 could also affect the cellular uptake. However, we believe these factors play a minor role in the differences seen in the cellular uptake because all of the micelles have similar average size. In fact, the spherical shape of the copolymer 3−DOX micelles should increase the uptake amount because their low aspect ratio should increase the uptake rate from the point of view of cellular membrane retraction. Thus, the low uptake of the copolymer 3−DOX 3770
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Figure 4. Confocal laser scanning microscopy images showing the MCF-7 cellular uptake of free DOX (A−C), copolymer 1−DOX micelles (D−F), copolymer 2−DOX micelles (G−I), and copolymer 3−DOX micelles (J−L) following 0.5 h of exposure. Panels A, D, G, and J show the location of the DOX (red), panels B, E, H, and K show the location of the Hoechst 33342 stained cell nucleus (blue), and panels C, F, I, and L show the overlay of DOX and Hoechst 33342 channels. The image exposure time of the free DOX was lowered due to its overwhelming signal strength of DOX, while that of the conjugates was identical. The scale bar represents 20 μm.
with the copolymer 3−DOX micelles showed a 10% increase in body weight, which was identical to the control mice injected with physiological saline. However, the body weight of mice injected with free DOX and the copolymer 2−DOX micelles
kg every day or two days as indicated by the arrows in Figure 6B and compared with mice treated with physiological saline as the control, and the tumor volume and mouse body weight were monitored. After eight days of treatment, mice treated 3771
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The free DOX and the copolymer 2−DOX micelles showed slightly better antitumor efficacy after 8 days, but the tumor growth rates found for these two groups were not lower than that of the mice treated with the copolymer 3−DOX micelles until the mice began to show clear weight loss. Hence, the lower tumor growth of the DOX and the copolymer 2−DOX micelle groups may be impacted by the severe systemic toxicity. One of the most important goals in the development of NDVs is to achieve therapeutic efficacy while minimizing the side effects and the systemic toxicity during treatment.43 In our work, only the copolymer 3−DOX micelles fulfill this goal. This outstanding property can be attributed to the prolonged circulation time which likely leads to enhanced accumulation in the tumor tissue44 and less accumulation in other untargeted organs and tissues. The results of this study indicate the necessity of introducing a negatively charged component to neutralize the positive charge of basic drugs or basic functional moieties conjugated to charge-balanced zwitterionic polymer drug delivery systems. The positive charge accumulation on the zwitterionic polymerbased NDV caused severe protein adsorption, fast cell internalization, short circulation time, and high acute toxicity that led to the rapid death of mice. The inclusion of excess negative SMA in the CB-based NDVs prolonged the circulation
Figure 5. Mean ± SD (N = 3) of the normalized COS-7 cell viability levels following incubation for 24 h with free DOX or polymer−DOX micelles as measured with an MTT assay. The results are normalized to the cell viability of COS-7 cells that were not exposed to DOX (100% viability).
decreased more than 10% as shown in Figure 6C. A 55% tumor-inhibition rate (TIR) was found for the mice treated with the copolymer 3−DOX micelles as shown in Figure 6B.
Figure 6. Mean ± SD (N = 5) of the (A) blood clearance of free DOX·HCl and the polymer−DOX micelles in female ICR mice, (B) normalized in vivo tumor inhibition caused by DOX·HCl and polymer−DOX micelles in female BALB/c mice, and (C) normalized change in mice body weight during the tumor treatment. In the tumor inhibition experiments the dosage dates are indicated by the arrows and the original tumor volume and mouse body weights before treatment were set as 100%. 3772
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time and demonstrated effective antitumor activity without causing any obvious systemic toxicity. These results indicate the importance of controlling the surface charge of zwitterionic polymer-based NDV. It is believed that a fine-tuning of the surface charge of the zwitterionic polymer-based NDV can be accomplished to achieve even better performance. In addition, NDVs with different surface charge densities or charge providing groups can lead to pH-responsive surface charges.45 This is a unique advantage to PEG-based NDVs, because the surface charge of these systems is determined by the intrinsic properties of PEG and charge shielding, and these are not easily altered.
4. CONCLUSIONS In this work, we demonstrated an approach to neutralize the positive charge imparted to a NDV by a conjugated basic drug (DOX) and the residue functional group (hydrazine group) on a charge-balanced zwitterionic polymer (pCBMA)-based system by including a negatively charged monomer (SMA). Polymer−DOX micelles with different surface charges were prepared and evaluated. By introducing the SMA component, the NDV with a slightly negative surface charge achieved the best circulation times and good anticancer therapeutic efficacy without causing obvious systemic toxicity. The positively charged and neutral NDVs performed poorly or induced severe systemic toxicity. These results indicate that chargebalanced zwitterionic polymer-based NDV face the challenge of maintaining overall charge balance after linkage with positively charged drugs or functional groups. The surface charge of the carrier is easily altered and leads to high toxicity when there are extra positively charged drugs or functional groups. The inclusion of an extra negatively charged component is required to tune the NDV surface charge, leading to a safer carrier with anticancer properties and low systemic toxicity.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors appreciate financial support from the National Nature Science Foundation of China (20974095, 20936005, 21174127, 31350110223), the Ph.D. Programs Foundation of Ministry of Education of China (20110101110034) and Zhejiang Provincial Natural Science Foundation of China (LZ13E030001).
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