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Drug Delivery
Tumor-Triggered Controlled Drug Release from Electrospun Fibers Using Inorganic Caps for Inhibiting Cancer Relapse Xin Zhao, Ziming Yuan, Lara Yildirimer, Jingwen Zhao, Zhi Yuan (William) Lin, Zhi Cao, Guoqing Pan, and Wenguo Cui* Cancer remission is characterized by a disappearance of signs and symptoms of cancer. Individual cancer cells, however, may still reside within the body representing a potential reservoir for cancer recurrence. Return of cancer after treatment (cancer recurrence) cannot be readily detected after a period of time until the size of tumor is developed to 1 cm. Direct targeting and destruction of tumors of undetectable sizes is therefore believed to not only relieve adverse symptoms resulting from cancer cells but also ensure complete recovery of the body to the premorbid state. Cancer cells are known to secrete acids which reduce the pH within and around the tumor tissue from 7.4 (normal tissue) to below 6.8 (pathological tissue). Taking this as an opportunity, the design and development of localized drug delivery devices that release chemotherapeutic medications upon stimulation via pH changes is hypothesized to treat cancer recurrence. Electrospun polymer fibers possess distinctive structural characteristics that mimic the nanoscale properties of native extracellular matrix, high drug loading capacities, and tunable release properties, which have garnered significant Dr. X. Zhao, J. Zhao, Z. Y. (William) Lin, Dr. G. Pan, Prof. W. Cui Department of Orthopedics The First Affiliated Hospital of Soochow University Orthopedic Institute Soochow University 708 Renmin Road, Suzhou, Jiangsu 215006, P. R. China E-mail: [email protected] Z. Yuan Department of General Surgery Shanghai Sixth People’s Hospital Shanghai Jiao Tong University School of Medicine 600 Yishan Road, Shanghai 200233, P. R. China Dr. L. Yildirimer Centre for Nanotechnology and Regenerative Medicine UCL Division of Surgery and Interventional Science University College London London WC1E 6AU, UK Dr. Z. Cao Department of Chemistry University of California Berkeley, CA 94720, USA DOI: 10.1002/smll.201500985
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interests for cancer treatment due to the capabilities of suppressing tumor cells and simultaneously supporting normal tissue regeneration at tumor resection sites.[1,2] There has also been an increasing interest to create stimuli-sensitive drug delivery systems by combining electrospinning technology with stimuli-responsive “gatekeepers.”[3] Drug release from such “gatekeepers” is induced only when stimuli including temperature,[4] pH,[5] light,[6] ultrasound,[7] and electric/magnetic field[8] are applied, and thus minimizes cytotoxic effects of drugs to healthy tissues and preserves bioactivity of loaded drugs. As mentioned above, utilizing a pH change as a drugrelease trigger is most appealing in cancer therapy due to the natural acidic property of tumor tissues.[9,10] The place and time to deploy a drug in such “gated” systems can be regulated via pH changes, which is therefore considered a fundamental concept for achieving highly specific and accurate anticancer drug delivery.[11] So far, pH responsive fibrous biomaterials have shown to release drugs from a few minutes to a few days via the mechanism of either protonation/deprotonation balance[5,12,13] or hydrolysis of polymers.[14,15] However, these systems may suffer from initial burst release or drug release at normal pH, leading to a biological damage to normal cells. Particularly at the initial healing stage after tumor resection, as few residual tumor cells are present, burst release of large quantity of drugs may result in decreased production of blood cells, lower immunity against bacterial infection, and even death resulting from diseases such as refractory pneumonia.[16] Furthermore, the fiber structure may collapse during drug release, making it difficult to support tissue regeneration. As tumor treatment is a long-lasting process, we thus believe that it is a promising strategy in cancer therapy to employ electrospun polymer fibers with acidic pH-triggered “caps” that exhibit localized long-term drug release to kill tumor cells without biological damage to normal cells while maintaining their structural integrity to support tissue regeneration. Previously, our group has developed electrospun poly(llactide) (PLLA) fibrous tissue engineering scaffolds with long-term structure integrity into which mesoporous silica nanoparticles (MSNs) were incorporated to prolong the drug release with tunable release kinetics.[1] However, drug release from this system is not triggered by acidic pH. Recent studies have reported the functionalization of MSNs with
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pH-responsive caps using polymer unit, including supramolecular nanovalves, pH-sensitive linkers, and polyelectrolytes, etc, to control the opening of pore entrances of MSNs to regulate the release of encapsulated drugs in the MSNs.[17–24] Nevertheless, when these MSNs are incorporated into polymer fibers, the surface macromolecules can hardly extend or degrade and thus may not allow the opening of MSN entrances and drug diffusion as the amount of surrounding water is low, which presents a hindrance for triggered drug release from MSNs upon stimulation of pH. Instead of organic “caps,” in this communication, we present the idea of using pH-triggered inorganic “caps” with CO32− functional groups on MSNs in electrospun fibers. When the electrospun PLLA fibers with CO32− functionalized MSNs encounter an acidic environment, the inorganic species immediately reacts to produce CO2 gas, which may accelerate the penetration of water to the inner cores of the PLLA nanofibers and facilitate the drug release. Such ion reactions of inorganic “caps” may circumvent the challenges of limited reaction/opening of organic gates on MSNs in low liquid environment inside the PLLA fibers. In this study, we report the construction of a smart tumortriggered controlled drug release system incorporated into electrospun fibers using inorganic caps for the eradication of cancer cells. As a proof of concept, we implanted this system into a mouse liver cancer model to verify its potential application in cancer relapse treatment. CaCO3 was used as the inorganic cap to control the opening of pore entrances of MSNs encapsulated inside PLLA fibers.[25] The primary motivations for employing CaCO3 lie in the fact that it is easily fabricated and inexpensive. Further, CaCO3 is stable at physiological pH of 7.4 while readily dissolving into biocompatible Ca2+ (cations) and CO2 gas in response to an acidic environment (pH < 6.8) which is frequently encountered in and around cancer tissues.[26] The hypothesized working principle of the pH responsive controlled-release system is illustrated in Figure 1a. The anticancer drug doxorubicin (DOX) was physically adsorbed onto MSNs and passively diffused through the mesoporous channels into the cores of the MSNs. Once the drug was loaded, channels were physically blocked by reacting CaCl2 lining the channels with supersaturated Na2CO3 to precipitate solid CaCO3.[25] This simple reaction has provided a facile route to construct CaCO3 gates. When the protons (H+) from the acids released by the tumor cells infiltrate into the electrospun PLLA fibers, H+ reacts with the CaCO3 to generate CO2 gas, enabling water penetration into the PLLA fibers thus facilitating the release of the anticancer drug. We conceived that such CaCO3 capped MSN system could spontaneously switch on drug release depending on local pH condition (i.e., neutral pH under normal condition versus acidic pH during tumor recurrence). The electrospun PLLA fibers with intelligent drug carriers should hence be capable of pHresponsive drug release in order to maximize therapeutic efficacy and to minimize undesired side effects, while serving as a tissue engineering scaffold. The uniformly spherical morphology of typical MSNs was observed in the scanning electron microscopy (SEM) image (Figure S1a, Supporting Information). The particle small 2015, 11, No. 34, 4284–4291
size distribution was demonstrated to cluster between 50 and 150 nm with mean particle diameter of ≈90 nm (Figure S1b, Supporting Information). Transmission electron microscopy (TEM) images (Figure 1b) revealed that the obtained MSNs possessed a porous and well-defined structure.[27] The anticancer DOX was loaded as a guest molecule by soaking MSNs in deionized water (pH 7.4) saturated with DOX (MSN-DOX). The MSN-DOX were then immersed subsequently in CaCl2 and Na2CO3 solution to precipitate solid CaCO3. After CaCO3 precipitation, the MSNs appeared more opaque (Figure 1c), with a surface area reduction from 1110 to 174 m2 g−1, reduced pore volume from 2.20 to 1.14 m2 g−1 and pore size from 3.17 to 2.57 nm (Table S1, Supporting Information), indicative of successful CaCO3 precipitation. The amount of DOX and CaCO3 loaded in 200 mg MSNs were measured by UV spectroscopy and ion chromatography, respectively, and found to be 5.6 and 57 mg. To investigate the gating behavior of MSN-CaCO3 systems, release experiments were performed at varying pH values. According to Figure 1d, at any given time point, the amount of DOX released from CaCO3-capped MSNs increased as pH decreased. At physiological pH (7.4), DOX release from MSNs without CaCO3 gates was revealed to be 100% over a 100 h incubation period. In contrast, DOX release from MSN-DOX-CaCO3 was dramatically lower, only 10% in 100 h, indicating that DOX was localized within MSN pores due to CaCO3 blocking drug diffusion routes. When the immersion medium was reduced to pH 5.0 and even 3.0 (more H+ available), an significantly increased release of DOX was seen (40% at pH 5.0 and 100% at pH 3.0 in 100 h), consistent with increased Ca2+ release (as a proxy measure of CaCO3 breakdown) (Figure S2, Supporting Information). These results have demonstrated that the fast uncapping response was on the basis of the decomposition of CaCO3 in a pH dependent manner, making it suitable as a switch for drug release.[25] To examine the antitumor effect of released DOX, HeLa cells which are well-known for their sensitivity to DOX were used as cancerous cell models.[28] MSNs in the absense or presence of CaCO3 were soaked in culture medium for 24 h at different pHs and the conditioned medium containing released DOX were used to culture HeLa cells. As shown in Figure 1e, the DNA concentrations of cells incubated with MSN-DOX at physiological pH was close to zero while cells were viable and continued to proliferate when exposed to CaCO3 capped MSN-DOX. This result was ascribed to the amount of drug released: at pH 7.4, only small amounts of cytotoxic DOX was released in the presence of CaCO3 gate leading to comparable cell viability to that on tissue culture plastic (TCP) whereas in absence of CaCO3 gate, large amount of DOX was released, resulting in ultimate cell death. The small amount of DOX released from the capped MSN systems may be due to some DOX molecules being absorbed onto the MSN outer surface to be instantly released upon contact with any solution and had resulted in small changes in cell morphology from spindle shape on TCP to a slightly rounded coutour (Figure S3, Supporting Information). In more acidic environments (i.e., pH 5.0 and 3.0), the acids triggered the opening of the CaCO3 gates, enabling
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Figure 1. a) Schematic illustration of construction and release mechanism of pH-responsive controlled release system. DOX adsorbed to MSN pores was entrapped by end-capping with inorganic CaCO3. The subsequent release of DOX was initiated by an effective displacement reaction in the presence of protons (H+). b,c) TEM images of MSNs before and after gating of channels, respectively. d) Time course of DOX release from MSN-DOX in the absence (black line) and presence (purple, blue, red lines) of CaCO3 gates and at different pHs. e) Proliferation of HeLa cells cultured in medium conditioned by DOX released from MSN-DOX-CaCO3 at different pHs. * correspond to P < 0.05 compared with control TCP.
rapid release of DOX which substantially reduced the viability of cancerous cells (Figure 1e and Figure S3, Supporting Information). The above results have clearly confirmed a highly effective pH-operable drug-release behavior of MSN-CaCO3. Electrospun fibrous scaffolds can provide a favorable microenvironment for tissue reconstruction after tumor resection.
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Thus, in order to prepare electrospun fibers capable of pH-triggered controlled drug release, PLLA was first dissolved in a solvent mixture composed of dichloromethane (DCM), ammonium fluoride, hexafluoroisopropanol (HFIP), and ethanol (EtOH). MSN-DOX or MSN-DOX-CaCO3 were separately dispersed in DCM by sonication. Vigorous stirring of the PLLA solution with the MSN suspension
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Figure 2. SEM a–c) and TEM d–f) images of PLLA fibers co-electrospun with (a, d) DOX, (b, e) MSN-DOX, and (c, f) MSN-DOX-CaCO3. EDX spectra of g) PLLA-DOX, h) PLLA-MSN-DOX, and i) PLLA-MSN-DOX-CaCO3.
then yielded an electrospinnable solution. The electrospun PLLA fibers showed slightly increased surface roughness and significantly increased diameter with the addition of the MSNs, possibly due to the increased viscosity of the fiber solution upon addition of MSNs (see Figure 2, compare a, b, and c).[2] MSNs could be clearly identified and were uniformly dispersed inside the PLLA fibers, indicating even and stable drug release (Figure 2, d–f). The energy-dispersive X-ray spectroscopy (EDX) spectra of PLLA-MSN exhibited additional peaks at Si (1.78 keV) compared to PLLA, owing to the incorporation of MSNs (see example in Figure 2g,h). The PLLAMSN-CaCO3 spectra showed new peaks at Ca (3.71 keV) due to the capping of CaCO3 on MSNs. Successful incorporation of MSNs was demonstrated visually as well as chemically. To determine the influence of MSNs on the mechanical properties of PLLA fibrous mats, uniaxial tensile tests were performed on mats of PLLA-DOX, PLLA-MSN-DOX and PLLA-MSN-DOX-CaCO3. The results demonstrated that incorporation of MSNs did not influence the tensile strengths or moduli of the fibrous PLLA membranes significantly although it reduced the stain at break slightly (Figure S4, Supporting Information). To validate pH-triggered controlled cargo release of CaCO3-capped PLLA-MSN-DOX systems, release experiments were conducted at different pH values. In order to demonstrate the importance of pH-triggered gate mechanisms, free DOX, MSN-DOX, and MSN-DOX-CaCO3 were co-electropsun with PLLA, respectively and incubated at physiological (7.4) as well as acidic pH (5.0 or 3.0) (Figure 3). small 2015, 11, No. 34, 4284–4291
As expected, PLLA-DOX exhibited an initial burst release of DOX with subsequent loss of all incorporated DOX over a period of 40 d. At pH 7.4 and 5.0, no significant difference in release rate or pattern was observed, while DOX was slightly more rapidly lost from PLLA fibers at pH 3.0 possibly due to increased water solubility of DOX in acidic environment or faster polymer degradation.[29] DOX incorporation into MSNs and subsequent electrospinning with PLLA (PLLA-MSNDOX) demonstrated significantly reduced but constant DOX release over 40 d compared to PLLA-DOX alone (Figure S5, Supporting Information). This prolonged DOX release was ascribed to the extended diffusion route from MSNs to PLLA and from PLLA to surrounding environment.[1] However, neither PLLA-DOX nor PLLA-MSN-DOX demonstrated significant pH-dependent DOX release compared to PLLA-MSN-DOX-CaCO3 with pH-sensitive CaCO3 gates (Figure 3b). The H+-sensitive feature of CaCO3 is a distinctive advantage of the PLLA-MSN-DOX-CaCO3 system as it opens up new possibilities for a simple and sensitive cargo delivery. As demonstrated in Figure 3b, the closed state at physiological pH 7.4 strongly constrained the delivery of the cargo. This is of particular interest in postsurgical cancer treatment as a large amount of DOX in the absense of tumor recurrence may cause DOX-related acute cardiotoxicity and resistance of cancer cells.[30,31] In contrast, a distinct release of the entrapped DOX was triggered in the open state as a result of CaCO3 decomposition when the pH was reduced (Figure 3b). At pH 5.0, DOX release was restricted for the
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Figure 3. Release profile of DOX from electrospun a) PLLA and b) PLLA-MSN-DOX-CaCO3 at different pHs. c) Schematic illustration of release mechanism of pH-responsive controlled release system. When the protons (H+) from the acids infiltrate into the electrospun PLLA fibers, H+ reacts with the CaCO3 to unveil the capped DOX inside the MSN pores and generate CO2 gas, enabling water penetration into the PLLA fibers and facilitating DOX release.
initial 5 d. The slow liberation of DOX at early stage may be due to the slow acid penetration into the electrospun fibers and reaction with CaCO3. After 5 d of incubation, a sudden increase in DOX release to 20% was observed with continuous and stable release from 20% to 60% for the remaining 30 d. At pH 3.0, DOX release was restricted for only 2 d probably due to faster reaction at higher concentration of H+ prior to opening of the CaCO3 gate. After 2 d of incubation, a dramatical increase in DOX release to 60% at day 10 was observed with continuous and stable release from 60% to 100% for the rest 30 d. The more rapid delivery of cargo from PLLA-MSN-DOX-CaCO3 compared to PLLA-MSN-DOX may be attributed to the formation of CO2 gas during the reaction between CaCO3 and H+, which enables water penetration into the fiber which, in turn, accelerates drug release. Interestingly, for PLLA-MSN-DOX-CaCO3, even at pH 3.0, there was steady release of DOX which lasted throughout the incubation time of 40 d, indicating that drug delivery using our PLLA-MSN-DOX-CaCO3 system may be extremely long-lived and continuous at both moderate and highly acidic pH. We concluded the working principle of DOX release in Figure 3c: when the electrospun PLLA fibers with capped MSNs encountered acidic environment, the CaCO3 gate would immediately react to produce CO2 gas, which enables water penetration into the fiber core, thereby facilitating fast drug release. To study the antitumor effect of the electrospun PLLADOX, PLLA-MSN-DOX, and PLLA-MSN-DOX-CaCO3 fibers at different pHs over different release period, after
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fiber soaking for 5, 20, and 40 d for the release study, they were removed from the release medium and immersed in culture medium for 24 h. The culture media containing the released DOX from these formulations were subsequently incubated with HeLa cells. As shown in Figure S6, Supporting Information, the DNA concentrations of cells incubated with PLLA-DOX at all pHs was close to zero after release for 5 d while cells were viable and continued to proliferate after release for 20 and 40 d, respectively, corresponding to great amount of DOX released initially but insufficient at later stages against cancerous HeLa cells. When the DOX were loaded in MSNs, at all pHs, as the drug diffusive route was extended, the drug release rate was reduced with prolonged release period, leading to continuous DOX release throughout the study period with cytotoxic activities to HeLa cells. When the MSNs were further gated with CaCO3, their cytotoxicity, however, showed the pH-dependent manner against HeLa cells. It was evident that the cytotoxicity of released DOX for HeLa cells was enhanced with reduction in pH. At physiological pH, as CaCO3 was insoluble in water, they blocked the opening of the MSNs and restricted the release of DOX, leading to non-cytotoxicity against HeLa cells at all stages with comparable cell proliferation (Figure S6, Supporting Information) and morphology (see example in Figure S7, Supporting Information) to TCP. Nevertheless, when the PLLA-MSN-DOX-CaCO3 electrospun fibers encountered acidic solutions, the acids triggered the opening of the CaCO3 gates, enabling rapid release of DOX which substantially reduced the viability of tumor cells for
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Figure 4. a) Representative images of H&E stained slices of tumor tissues after 4 and 8 weeks of exposure to PLLA-DOX, PLLA-MSN-DOX or PLLAMSN-DOX-CaCO3. The red arrows indicate areas of necrosis resulting from sustained DOX release. b) Immunohistochemical slices of tumor tissue stained for caspase-3 - a marker for cellular apoptosis. c) Caspase-3 activity and d) extent of apoptosis at 4 and 8 weeks in response to exposure to PLLA-DOX, PLLA-MSN-DOX, and PLLA-MSN-DOX-CaCO3. * correspond to P < 0.05 compared with PLLA-MSN-DOX-CaCO3.
the whole study period. Clearly, pH (concentration of H+) played a decisive role in DOX release from the electrospun PLLA-MSN-CaCO3 fibers, which was consistent with the observations of Figure 3b. These results suggested that the MSNs could be an effective drug carrier for extended release period from PLLA fibers and DOX could be encapsulated in MSNs without release until the CaCO3 caps on MSNs was removed by reaction of CaCO3 with H+ encountered in tumor environment. To realize the potential application in cancer therapy of our system as pH-switchable electrospun fibers for drug release, subcutaneous implantation of different PLLA fibers (i.e., PLLA-DOX, PLLA-MSN-DOX, and PLLA-MSNDOX-CaCO3) were performed in constructed mouse liver model. We first examined the tumor response to different electrospun fibers over 8 weeks. As can be seen in the hematoxylin and eosin (H&E) images in Figure 4a, implantation of PLLA-MSN-DOX-CaCO3 scaffolds onto tumor tissues resulted in increasing areas of necrosis over a period of 8 weeks, consistent with sustained DOX release (Figure 3b). After 4 weeks, small areas of necrotic tumor tissues could be seen in both PLLA-DOX and PLLA-MSN-DOX-CaCO3 group possibly due to the initial fast release of DOX. In the PLLA-MSN-DOX group, necrotic tumor tissues were barely observed possibly due to the slow release of DOX. After 8 weeks, the PLLA-DOX group demonstrated reduced areas small 2015, 11, No. 34, 4284–4291
of necrotic tissue likely due to the depletion of DOX. In the PLLA-MSN-DOX and PLLA-MSN-DOX-CaCO3 groups, areas of necrotic tumor tissue could be readily identified with areas of necrosis consistently being the largest in the PLLAMSN-DOX-CaCO3 groups. In the controls with no implanted scaffolds, necrotic tumor tissues could not be readily identified although they appeared occasionally possibly due to the nutritional depletion during the tumor development. These results futher confirmed that the closed CaCO3 gate in the PLLA fibrous system could be opened and allow release of encapsulated DOX in the tumor site with acids secreted by tumor cells.[32] Moreover, the presence of MSNs to prolong the drug release has endowed the system with continuous inhibition of tumor development over the 8 week study period. Caspase 3, an apoptosis-related cysteine peptidase, was further used to confirm the apoptotic activity of cancer cells in present study.[33] As shown in the immunohistochemical images in Figure 4b, abundant caspase-3 activity signifying apoptosis of cancer cells could be observed in all but the control samples. The rate of caspase actvity correlated well with the release pattern of DOX in the different PLLA-MSNDOX systems. Caspase-3 activity and the number of apoptotic cells of PLLA-DOX group increased rapidly throughout the first 4 weeks but then slowed down at 8 weeks, likely due to the initial burst release of DOX and subsequent depletion
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at later stages. Caspase-3 activity and the number of apoptotic cells of the PLLA-MSN-DOX group at the first 4 weeks were lowest compared to other groups possibly due to insufficient release of DOX but they increased at 8 weeks owing to the increased DOX release at later stages. Caspase activity and the number of apoptotic cells of the PLLA-MSN-DOXCaCO3 group, on the other hand, increased continuously throughout the 8 week study period and was demonstrably the highest compared to all other groups. The above results revealed that the drug delivery system of PLLA-MSN-DOXCaCO3 displayed the highest capability to induce cancer cell apoptosis and inhibit tumor recurrence. This system possessed not only pH responsiveness but also a sustained release pattern of DOX. Such a long-lasting release profile was in accordance with the double barrier consisting of the CaCO3 gate and PLLA coating which prolonged the release of DOX over an extended period of time. In this study, we report the fabrication of a novel and facile pH-triggered controlled release system using inorganic cap CaCO3 to control the opening of pore entrances of drug loaded MSNs inside PLLA fibers. We found that this unique class of smart carriers could promptly unload the encapsulated drug upon stimulation of acidic pH as well as provide sustained release of DOX from the electrospun PLLA fibers. This system took advantage of cancer itself (acidic pH) to trigger the release of anticancer drug, demonstrating an effective example of disease-triggered cancer therapy.[3,34] In addition, this strategy may not only be a rescue of slow reaction/opening when using conventional organic gates on MSNs in absence of sufficient liquid inside the PLLA fibers, but also be of high sensitivity to acid stimuli with resultant CO2 gas to enable water penetration into the fiber which is due to the quick response of CaCO3 to acidic environment (CaCO3 + 2H+ → Ca2+ + H2O + CO2↑), further facilitating the drug release and resulting in a significant reduction in cancer cell viability over an extended period of time. More importantly, the reduced or inhibited drug release at normal pH may decrease side effects by preventing cytotoxic drugs from harming healthy tissues and by preserving bioactivity of loaded drugs. Furthermore, as known, the electrospun composite fibers can serve as a tissue engineering scaffold to support tissue regeneration after tumor resection.[35,36] This exceptional delivery system has thus advantages of localized drug delivery with high sensitivity to local environment even at low-liquid environment as well as prolonged release with tunable release kinetics, leading to potentially improved therapeutic efficacy against pathological tissues and reduced detrimental effect to normal tissues. In summary, we have demonstrated the facile assembly of a smart tumor-triggered controlled drug release system from electrospun PLLA-MSN composite fibers using highly sensitive inorganic CaCO3 cap. This ability to target release of drugs encapsulated within MSNs may prove beneficial in preempting cancer recurrence by eliminating tumor recurrence to begin with. This system avoided drug release at normal pH, thus limiting the risk of biological damage to normal cells but demonstrated the capacity for pH-triggered and sustained drug release over a period of 40 d. Due to the high sensitivity of the inorganic calcium gates
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to environmental pH changes, the drug release profile was dependant on the content of hydrogen ions. More importantly, the released drugs exhibited prolonged in vitro and in vivo antitumor efficacy which lasted throughout the study period. The authors envision that this new delivery vehicle can find widespread applications in cancer therapy to provide sustained drug release to eliminate cancerous tissues (acidic environment) while restricting drug release to avoid damage to normal tissues (physiological pH).
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements X.Z. and Z.Y. contributed equally to this work. This work was supported by the National Natural Science Foundation of China (51373112 and 51003058), Jiangsu Provincial Special Program of Medical Science (BL2012004), Jiangsu Provincial Clinical Orthopedic Center, the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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[28] T. K. Lee, T. C. Lau, I. O. Ng, Cancer Chemother. Pharmacol. 2002, 49, 78. [29] L. Zhang, J. Xia, Q. Zhao, L. Liu, Z. Zhang, Small 2010, 6, 537. [30] F. Tewes, E. Munnier, B. Antoon, L. Ngaboni Okassa, S. Cohen-Jonathan, H. Marchais, L. Douziech-Eyrolles, M. Soucé, P. Dubois, I. Chourpa, Eur. J. Pharm. Biopharm. 2007, 66, 488. [31] S. Cai, S. Thati, T. R. Bagby, H. M. Diab, N. M. Davies, M. S. Cohen, M. L. Forrest, J. Controlled Release 2010, 146, 212. [32] H. Shime, M. Yabu, T. Akazawa, K. Kodama, M. Matsumoto, T. Seya, N. Inoue, J. Immunol. 2008, 180, 7175. [33] B. T. Hyman, J. Yuan, Nat. Rev. Neurosci. 2012, 13, 395. [34] X. Zhao, W. Cui, Mater. Today 2014, 18, 56. [35] S. Zhao, X. Zhao, S. Dong, J. Yu, G. Q. Pan, Y. Zhang, J. Z. Zhao, W. G. Cui, J. Mater. Chem. B 2015, 3, 990. [36] X. Zhao, J. W. Zhao, Z. Y. Lin, G. Q. Pan, Y. Q. Zhu, Y. S. Cheng, W. G. Cui, Colloids Surf. B 2015, 130, 1.
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Drug Delivery
Tumor-Triggered Controlled Drug Release from Electrospun Fibers Using Inorganic Caps for Inhibiting Cancer Relapse Xin Zhao, Ziming Yuan, Lara Yildirimer, Jingwen Zhao, Zhi Yuan (William) Lin, Zhi Cao, Guoqing Pan, and Wenguo Cui* Cancer remission is characterized by a disappearance of signs and symptoms of cancer. Individual cancer cells, however, may still reside within the body representing a potential reservoir for cancer recurrence. Return of cancer after treatment (cancer recurrence) cannot be readily detected after a period of time until the size of tumor is developed to 1 cm. Direct targeting and destruction of tumors of undetectable sizes is therefore believed to not only relieve adverse symptoms resulting from cancer cells but also ensure complete recovery of the body to the premorbid state. Cancer cells are known to secrete acids which reduce the pH within and around the tumor tissue from 7.4 (normal tissue) to below 6.8 (pathological tissue). Taking this as an opportunity, the design and development of localized drug delivery devices that release chemotherapeutic medications upon stimulation via pH changes is hypothesized to treat cancer recurrence. Electrospun polymer fibers possess distinctive structural characteristics that mimic the nanoscale properties of native extracellular matrix, high drug loading capacities, and tunable release properties, which have garnered significant Dr. X. Zhao, J. Zhao, Z. Y. (William) Lin, Dr. G. Pan, Prof. W. Cui Department of Orthopedics The First Affiliated Hospital of Soochow University Orthopedic Institute Soochow University 708 Renmin Road, Suzhou, Jiangsu 215006, P. R. China E-mail: [email protected] Z. Yuan Department of General Surgery Shanghai Sixth People’s Hospital Shanghai Jiao Tong University School of Medicine 600 Yishan Road, Shanghai 200233, P. R. China Dr. L. Yildirimer Centre for Nanotechnology and Regenerative Medicine UCL Division of Surgery and Interventional Science University College London London WC1E 6AU, UK Dr. Z. Cao Department of Chemistry University of California Berkeley, CA 94720, USA DOI: 10.1002/smll.201500985
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interests for cancer treatment due to the capabilities of suppressing tumor cells and simultaneously supporting normal tissue regeneration at tumor resection sites.[1,2] There has also been an increasing interest to create stimuli-sensitive drug delivery systems by combining electrospinning technology with stimuli-responsive “gatekeepers.”[3] Drug release from such “gatekeepers” is induced only when stimuli including temperature,[4] pH,[5] light,[6] ultrasound,[7] and electric/magnetic field[8] are applied, and thus minimizes cytotoxic effects of drugs to healthy tissues and preserves bioactivity of loaded drugs. As mentioned above, utilizing a pH change as a drugrelease trigger is most appealing in cancer therapy due to the natural acidic property of tumor tissues.[9,10] The place and time to deploy a drug in such “gated” systems can be regulated via pH changes, which is therefore considered a fundamental concept for achieving highly specific and accurate anticancer drug delivery.[11] So far, pH responsive fibrous biomaterials have shown to release drugs from a few minutes to a few days via the mechanism of either protonation/deprotonation balance[5,12,13] or hydrolysis of polymers.[14,15] However, these systems may suffer from initial burst release or drug release at normal pH, leading to a biological damage to normal cells. Particularly at the initial healing stage after tumor resection, as few residual tumor cells are present, burst release of large quantity of drugs may result in decreased production of blood cells, lower immunity against bacterial infection, and even death resulting from diseases such as refractory pneumonia.[16] Furthermore, the fiber structure may collapse during drug release, making it difficult to support tissue regeneration. As tumor treatment is a long-lasting process, we thus believe that it is a promising strategy in cancer therapy to employ electrospun polymer fibers with acidic pH-triggered “caps” that exhibit localized long-term drug release to kill tumor cells without biological damage to normal cells while maintaining their structural integrity to support tissue regeneration. Previously, our group has developed electrospun poly(llactide) (PLLA) fibrous tissue engineering scaffolds with long-term structure integrity into which mesoporous silica nanoparticles (MSNs) were incorporated to prolong the drug release with tunable release kinetics.[1] However, drug release from this system is not triggered by acidic pH. Recent studies have reported the functionalization of MSNs with
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pH-responsive caps using polymer unit, including supramolecular nanovalves, pH-sensitive linkers, and polyelectrolytes, etc, to control the opening of pore entrances of MSNs to regulate the release of encapsulated drugs in the MSNs.[17–24] Nevertheless, when these MSNs are incorporated into polymer fibers, the surface macromolecules can hardly extend or degrade and thus may not allow the opening of MSN entrances and drug diffusion as the amount of surrounding water is low, which presents a hindrance for triggered drug release from MSNs upon stimulation of pH. Instead of organic “caps,” in this communication, we present the idea of using pH-triggered inorganic “caps” with CO32− functional groups on MSNs in electrospun fibers. When the electrospun PLLA fibers with CO32− functionalized MSNs encounter an acidic environment, the inorganic species immediately reacts to produce CO2 gas, which may accelerate the penetration of water to the inner cores of the PLLA nanofibers and facilitate the drug release. Such ion reactions of inorganic “caps” may circumvent the challenges of limited reaction/opening of organic gates on MSNs in low liquid environment inside the PLLA fibers. In this study, we report the construction of a smart tumortriggered controlled drug release system incorporated into electrospun fibers using inorganic caps for the eradication of cancer cells. As a proof of concept, we implanted this system into a mouse liver cancer model to verify its potential application in cancer relapse treatment. CaCO3 was used as the inorganic cap to control the opening of pore entrances of MSNs encapsulated inside PLLA fibers.[25] The primary motivations for employing CaCO3 lie in the fact that it is easily fabricated and inexpensive. Further, CaCO3 is stable at physiological pH of 7.4 while readily dissolving into biocompatible Ca2+ (cations) and CO2 gas in response to an acidic environment (pH < 6.8) which is frequently encountered in and around cancer tissues.[26] The hypothesized working principle of the pH responsive controlled-release system is illustrated in Figure 1a. The anticancer drug doxorubicin (DOX) was physically adsorbed onto MSNs and passively diffused through the mesoporous channels into the cores of the MSNs. Once the drug was loaded, channels were physically blocked by reacting CaCl2 lining the channels with supersaturated Na2CO3 to precipitate solid CaCO3.[25] This simple reaction has provided a facile route to construct CaCO3 gates. When the protons (H+) from the acids released by the tumor cells infiltrate into the electrospun PLLA fibers, H+ reacts with the CaCO3 to generate CO2 gas, enabling water penetration into the PLLA fibers thus facilitating the release of the anticancer drug. We conceived that such CaCO3 capped MSN system could spontaneously switch on drug release depending on local pH condition (i.e., neutral pH under normal condition versus acidic pH during tumor recurrence). The electrospun PLLA fibers with intelligent drug carriers should hence be capable of pHresponsive drug release in order to maximize therapeutic efficacy and to minimize undesired side effects, while serving as a tissue engineering scaffold. The uniformly spherical morphology of typical MSNs was observed in the scanning electron microscopy (SEM) image (Figure S1a, Supporting Information). The particle small 2015, 11, No. 34, 4284–4291
size distribution was demonstrated to cluster between 50 and 150 nm with mean particle diameter of ≈90 nm (Figure S1b, Supporting Information). Transmission electron microscopy (TEM) images (Figure 1b) revealed that the obtained MSNs possessed a porous and well-defined structure.[27] The anticancer DOX was loaded as a guest molecule by soaking MSNs in deionized water (pH 7.4) saturated with DOX (MSN-DOX). The MSN-DOX were then immersed subsequently in CaCl2 and Na2CO3 solution to precipitate solid CaCO3. After CaCO3 precipitation, the MSNs appeared more opaque (Figure 1c), with a surface area reduction from 1110 to 174 m2 g−1, reduced pore volume from 2.20 to 1.14 m2 g−1 and pore size from 3.17 to 2.57 nm (Table S1, Supporting Information), indicative of successful CaCO3 precipitation. The amount of DOX and CaCO3 loaded in 200 mg MSNs were measured by UV spectroscopy and ion chromatography, respectively, and found to be 5.6 and 57 mg. To investigate the gating behavior of MSN-CaCO3 systems, release experiments were performed at varying pH values. According to Figure 1d, at any given time point, the amount of DOX released from CaCO3-capped MSNs increased as pH decreased. At physiological pH (7.4), DOX release from MSNs without CaCO3 gates was revealed to be 100% over a 100 h incubation period. In contrast, DOX release from MSN-DOX-CaCO3 was dramatically lower, only 10% in 100 h, indicating that DOX was localized within MSN pores due to CaCO3 blocking drug diffusion routes. When the immersion medium was reduced to pH 5.0 and even 3.0 (more H+ available), an significantly increased release of DOX was seen (40% at pH 5.0 and 100% at pH 3.0 in 100 h), consistent with increased Ca2+ release (as a proxy measure of CaCO3 breakdown) (Figure S2, Supporting Information). These results have demonstrated that the fast uncapping response was on the basis of the decomposition of CaCO3 in a pH dependent manner, making it suitable as a switch for drug release.[25] To examine the antitumor effect of released DOX, HeLa cells which are well-known for their sensitivity to DOX were used as cancerous cell models.[28] MSNs in the absense or presence of CaCO3 were soaked in culture medium for 24 h at different pHs and the conditioned medium containing released DOX were used to culture HeLa cells. As shown in Figure 1e, the DNA concentrations of cells incubated with MSN-DOX at physiological pH was close to zero while cells were viable and continued to proliferate when exposed to CaCO3 capped MSN-DOX. This result was ascribed to the amount of drug released: at pH 7.4, only small amounts of cytotoxic DOX was released in the presence of CaCO3 gate leading to comparable cell viability to that on tissue culture plastic (TCP) whereas in absence of CaCO3 gate, large amount of DOX was released, resulting in ultimate cell death. The small amount of DOX released from the capped MSN systems may be due to some DOX molecules being absorbed onto the MSN outer surface to be instantly released upon contact with any solution and had resulted in small changes in cell morphology from spindle shape on TCP to a slightly rounded coutour (Figure S3, Supporting Information). In more acidic environments (i.e., pH 5.0 and 3.0), the acids triggered the opening of the CaCO3 gates, enabling
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Figure 1. a) Schematic illustration of construction and release mechanism of pH-responsive controlled release system. DOX adsorbed to MSN pores was entrapped by end-capping with inorganic CaCO3. The subsequent release of DOX was initiated by an effective displacement reaction in the presence of protons (H+). b,c) TEM images of MSNs before and after gating of channels, respectively. d) Time course of DOX release from MSN-DOX in the absence (black line) and presence (purple, blue, red lines) of CaCO3 gates and at different pHs. e) Proliferation of HeLa cells cultured in medium conditioned by DOX released from MSN-DOX-CaCO3 at different pHs. * correspond to P < 0.05 compared with control TCP.
rapid release of DOX which substantially reduced the viability of cancerous cells (Figure 1e and Figure S3, Supporting Information). The above results have clearly confirmed a highly effective pH-operable drug-release behavior of MSN-CaCO3. Electrospun fibrous scaffolds can provide a favorable microenvironment for tissue reconstruction after tumor resection.
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Thus, in order to prepare electrospun fibers capable of pH-triggered controlled drug release, PLLA was first dissolved in a solvent mixture composed of dichloromethane (DCM), ammonium fluoride, hexafluoroisopropanol (HFIP), and ethanol (EtOH). MSN-DOX or MSN-DOX-CaCO3 were separately dispersed in DCM by sonication. Vigorous stirring of the PLLA solution with the MSN suspension
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Figure 2. SEM a–c) and TEM d–f) images of PLLA fibers co-electrospun with (a, d) DOX, (b, e) MSN-DOX, and (c, f) MSN-DOX-CaCO3. EDX spectra of g) PLLA-DOX, h) PLLA-MSN-DOX, and i) PLLA-MSN-DOX-CaCO3.
then yielded an electrospinnable solution. The electrospun PLLA fibers showed slightly increased surface roughness and significantly increased diameter with the addition of the MSNs, possibly due to the increased viscosity of the fiber solution upon addition of MSNs (see Figure 2, compare a, b, and c).[2] MSNs could be clearly identified and were uniformly dispersed inside the PLLA fibers, indicating even and stable drug release (Figure 2, d–f). The energy-dispersive X-ray spectroscopy (EDX) spectra of PLLA-MSN exhibited additional peaks at Si (1.78 keV) compared to PLLA, owing to the incorporation of MSNs (see example in Figure 2g,h). The PLLAMSN-CaCO3 spectra showed new peaks at Ca (3.71 keV) due to the capping of CaCO3 on MSNs. Successful incorporation of MSNs was demonstrated visually as well as chemically. To determine the influence of MSNs on the mechanical properties of PLLA fibrous mats, uniaxial tensile tests were performed on mats of PLLA-DOX, PLLA-MSN-DOX and PLLA-MSN-DOX-CaCO3. The results demonstrated that incorporation of MSNs did not influence the tensile strengths or moduli of the fibrous PLLA membranes significantly although it reduced the stain at break slightly (Figure S4, Supporting Information). To validate pH-triggered controlled cargo release of CaCO3-capped PLLA-MSN-DOX systems, release experiments were conducted at different pH values. In order to demonstrate the importance of pH-triggered gate mechanisms, free DOX, MSN-DOX, and MSN-DOX-CaCO3 were co-electropsun with PLLA, respectively and incubated at physiological (7.4) as well as acidic pH (5.0 or 3.0) (Figure 3). small 2015, 11, No. 34, 4284–4291
As expected, PLLA-DOX exhibited an initial burst release of DOX with subsequent loss of all incorporated DOX over a period of 40 d. At pH 7.4 and 5.0, no significant difference in release rate or pattern was observed, while DOX was slightly more rapidly lost from PLLA fibers at pH 3.0 possibly due to increased water solubility of DOX in acidic environment or faster polymer degradation.[29] DOX incorporation into MSNs and subsequent electrospinning with PLLA (PLLA-MSNDOX) demonstrated significantly reduced but constant DOX release over 40 d compared to PLLA-DOX alone (Figure S5, Supporting Information). This prolonged DOX release was ascribed to the extended diffusion route from MSNs to PLLA and from PLLA to surrounding environment.[1] However, neither PLLA-DOX nor PLLA-MSN-DOX demonstrated significant pH-dependent DOX release compared to PLLA-MSN-DOX-CaCO3 with pH-sensitive CaCO3 gates (Figure 3b). The H+-sensitive feature of CaCO3 is a distinctive advantage of the PLLA-MSN-DOX-CaCO3 system as it opens up new possibilities for a simple and sensitive cargo delivery. As demonstrated in Figure 3b, the closed state at physiological pH 7.4 strongly constrained the delivery of the cargo. This is of particular interest in postsurgical cancer treatment as a large amount of DOX in the absense of tumor recurrence may cause DOX-related acute cardiotoxicity and resistance of cancer cells.[30,31] In contrast, a distinct release of the entrapped DOX was triggered in the open state as a result of CaCO3 decomposition when the pH was reduced (Figure 3b). At pH 5.0, DOX release was restricted for the
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Figure 3. Release profile of DOX from electrospun a) PLLA and b) PLLA-MSN-DOX-CaCO3 at different pHs. c) Schematic illustration of release mechanism of pH-responsive controlled release system. When the protons (H+) from the acids infiltrate into the electrospun PLLA fibers, H+ reacts with the CaCO3 to unveil the capped DOX inside the MSN pores and generate CO2 gas, enabling water penetration into the PLLA fibers and facilitating DOX release.
initial 5 d. The slow liberation of DOX at early stage may be due to the slow acid penetration into the electrospun fibers and reaction with CaCO3. After 5 d of incubation, a sudden increase in DOX release to 20% was observed with continuous and stable release from 20% to 60% for the remaining 30 d. At pH 3.0, DOX release was restricted for only 2 d probably due to faster reaction at higher concentration of H+ prior to opening of the CaCO3 gate. After 2 d of incubation, a dramatical increase in DOX release to 60% at day 10 was observed with continuous and stable release from 60% to 100% for the rest 30 d. The more rapid delivery of cargo from PLLA-MSN-DOX-CaCO3 compared to PLLA-MSN-DOX may be attributed to the formation of CO2 gas during the reaction between CaCO3 and H+, which enables water penetration into the fiber which, in turn, accelerates drug release. Interestingly, for PLLA-MSN-DOX-CaCO3, even at pH 3.0, there was steady release of DOX which lasted throughout the incubation time of 40 d, indicating that drug delivery using our PLLA-MSN-DOX-CaCO3 system may be extremely long-lived and continuous at both moderate and highly acidic pH. We concluded the working principle of DOX release in Figure 3c: when the electrospun PLLA fibers with capped MSNs encountered acidic environment, the CaCO3 gate would immediately react to produce CO2 gas, which enables water penetration into the fiber core, thereby facilitating fast drug release. To study the antitumor effect of the electrospun PLLADOX, PLLA-MSN-DOX, and PLLA-MSN-DOX-CaCO3 fibers at different pHs over different release period, after
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fiber soaking for 5, 20, and 40 d for the release study, they were removed from the release medium and immersed in culture medium for 24 h. The culture media containing the released DOX from these formulations were subsequently incubated with HeLa cells. As shown in Figure S6, Supporting Information, the DNA concentrations of cells incubated with PLLA-DOX at all pHs was close to zero after release for 5 d while cells were viable and continued to proliferate after release for 20 and 40 d, respectively, corresponding to great amount of DOX released initially but insufficient at later stages against cancerous HeLa cells. When the DOX were loaded in MSNs, at all pHs, as the drug diffusive route was extended, the drug release rate was reduced with prolonged release period, leading to continuous DOX release throughout the study period with cytotoxic activities to HeLa cells. When the MSNs were further gated with CaCO3, their cytotoxicity, however, showed the pH-dependent manner against HeLa cells. It was evident that the cytotoxicity of released DOX for HeLa cells was enhanced with reduction in pH. At physiological pH, as CaCO3 was insoluble in water, they blocked the opening of the MSNs and restricted the release of DOX, leading to non-cytotoxicity against HeLa cells at all stages with comparable cell proliferation (Figure S6, Supporting Information) and morphology (see example in Figure S7, Supporting Information) to TCP. Nevertheless, when the PLLA-MSN-DOX-CaCO3 electrospun fibers encountered acidic solutions, the acids triggered the opening of the CaCO3 gates, enabling rapid release of DOX which substantially reduced the viability of tumor cells for
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Figure 4. a) Representative images of H&E stained slices of tumor tissues after 4 and 8 weeks of exposure to PLLA-DOX, PLLA-MSN-DOX or PLLAMSN-DOX-CaCO3. The red arrows indicate areas of necrosis resulting from sustained DOX release. b) Immunohistochemical slices of tumor tissue stained for caspase-3 - a marker for cellular apoptosis. c) Caspase-3 activity and d) extent of apoptosis at 4 and 8 weeks in response to exposure to PLLA-DOX, PLLA-MSN-DOX, and PLLA-MSN-DOX-CaCO3. * correspond to P < 0.05 compared with PLLA-MSN-DOX-CaCO3.
the whole study period. Clearly, pH (concentration of H+) played a decisive role in DOX release from the electrospun PLLA-MSN-CaCO3 fibers, which was consistent with the observations of Figure 3b. These results suggested that the MSNs could be an effective drug carrier for extended release period from PLLA fibers and DOX could be encapsulated in MSNs without release until the CaCO3 caps on MSNs was removed by reaction of CaCO3 with H+ encountered in tumor environment. To realize the potential application in cancer therapy of our system as pH-switchable electrospun fibers for drug release, subcutaneous implantation of different PLLA fibers (i.e., PLLA-DOX, PLLA-MSN-DOX, and PLLA-MSNDOX-CaCO3) were performed in constructed mouse liver model. We first examined the tumor response to different electrospun fibers over 8 weeks. As can be seen in the hematoxylin and eosin (H&E) images in Figure 4a, implantation of PLLA-MSN-DOX-CaCO3 scaffolds onto tumor tissues resulted in increasing areas of necrosis over a period of 8 weeks, consistent with sustained DOX release (Figure 3b). After 4 weeks, small areas of necrotic tumor tissues could be seen in both PLLA-DOX and PLLA-MSN-DOX-CaCO3 group possibly due to the initial fast release of DOX. In the PLLA-MSN-DOX group, necrotic tumor tissues were barely observed possibly due to the slow release of DOX. After 8 weeks, the PLLA-DOX group demonstrated reduced areas small 2015, 11, No. 34, 4284–4291
of necrotic tissue likely due to the depletion of DOX. In the PLLA-MSN-DOX and PLLA-MSN-DOX-CaCO3 groups, areas of necrotic tumor tissue could be readily identified with areas of necrosis consistently being the largest in the PLLAMSN-DOX-CaCO3 groups. In the controls with no implanted scaffolds, necrotic tumor tissues could not be readily identified although they appeared occasionally possibly due to the nutritional depletion during the tumor development. These results futher confirmed that the closed CaCO3 gate in the PLLA fibrous system could be opened and allow release of encapsulated DOX in the tumor site with acids secreted by tumor cells.[32] Moreover, the presence of MSNs to prolong the drug release has endowed the system with continuous inhibition of tumor development over the 8 week study period. Caspase 3, an apoptosis-related cysteine peptidase, was further used to confirm the apoptotic activity of cancer cells in present study.[33] As shown in the immunohistochemical images in Figure 4b, abundant caspase-3 activity signifying apoptosis of cancer cells could be observed in all but the control samples. The rate of caspase actvity correlated well with the release pattern of DOX in the different PLLA-MSNDOX systems. Caspase-3 activity and the number of apoptotic cells of PLLA-DOX group increased rapidly throughout the first 4 weeks but then slowed down at 8 weeks, likely due to the initial burst release of DOX and subsequent depletion
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at later stages. Caspase-3 activity and the number of apoptotic cells of the PLLA-MSN-DOX group at the first 4 weeks were lowest compared to other groups possibly due to insufficient release of DOX but they increased at 8 weeks owing to the increased DOX release at later stages. Caspase activity and the number of apoptotic cells of the PLLA-MSN-DOXCaCO3 group, on the other hand, increased continuously throughout the 8 week study period and was demonstrably the highest compared to all other groups. The above results revealed that the drug delivery system of PLLA-MSN-DOXCaCO3 displayed the highest capability to induce cancer cell apoptosis and inhibit tumor recurrence. This system possessed not only pH responsiveness but also a sustained release pattern of DOX. Such a long-lasting release profile was in accordance with the double barrier consisting of the CaCO3 gate and PLLA coating which prolonged the release of DOX over an extended period of time. In this study, we report the fabrication of a novel and facile pH-triggered controlled release system using inorganic cap CaCO3 to control the opening of pore entrances of drug loaded MSNs inside PLLA fibers. We found that this unique class of smart carriers could promptly unload the encapsulated drug upon stimulation of acidic pH as well as provide sustained release of DOX from the electrospun PLLA fibers. This system took advantage of cancer itself (acidic pH) to trigger the release of anticancer drug, demonstrating an effective example of disease-triggered cancer therapy.[3,34] In addition, this strategy may not only be a rescue of slow reaction/opening when using conventional organic gates on MSNs in absence of sufficient liquid inside the PLLA fibers, but also be of high sensitivity to acid stimuli with resultant CO2 gas to enable water penetration into the fiber which is due to the quick response of CaCO3 to acidic environment (CaCO3 + 2H+ → Ca2+ + H2O + CO2↑), further facilitating the drug release and resulting in a significant reduction in cancer cell viability over an extended period of time. More importantly, the reduced or inhibited drug release at normal pH may decrease side effects by preventing cytotoxic drugs from harming healthy tissues and by preserving bioactivity of loaded drugs. Furthermore, as known, the electrospun composite fibers can serve as a tissue engineering scaffold to support tissue regeneration after tumor resection.[35,36] This exceptional delivery system has thus advantages of localized drug delivery with high sensitivity to local environment even at low-liquid environment as well as prolonged release with tunable release kinetics, leading to potentially improved therapeutic efficacy against pathological tissues and reduced detrimental effect to normal tissues. In summary, we have demonstrated the facile assembly of a smart tumor-triggered controlled drug release system from electrospun PLLA-MSN composite fibers using highly sensitive inorganic CaCO3 cap. This ability to target release of drugs encapsulated within MSNs may prove beneficial in preempting cancer recurrence by eliminating tumor recurrence to begin with. This system avoided drug release at normal pH, thus limiting the risk of biological damage to normal cells but demonstrated the capacity for pH-triggered and sustained drug release over a period of 40 d. Due to the high sensitivity of the inorganic calcium gates
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to environmental pH changes, the drug release profile was dependant on the content of hydrogen ions. More importantly, the released drugs exhibited prolonged in vitro and in vivo antitumor efficacy which lasted throughout the study period. The authors envision that this new delivery vehicle can find widespread applications in cancer therapy to provide sustained drug release to eliminate cancerous tissues (acidic environment) while restricting drug release to avoid damage to normal tissues (physiological pH).
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements X.Z. and Z.Y. contributed equally to this work. This work was supported by the National Natural Science Foundation of China (51373112 and 51003058), Jiangsu Provincial Special Program of Medical Science (BL2012004), Jiangsu Provincial Clinical Orthopedic Center, the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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