Locally Deployable Nanofiber Patch for Sequential Drug Delivery in

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A Locally Deployable Nanofiber Patch for Sequential Drug Delivery in Treatment of Primary and Advanced Orthotopic Hepatomas Jiannan Li, Weiguo Xu, Di Li, Tongjun Liu, Yu Shrike Zhang, Jianxun Ding, and Xuesi Chen ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01729 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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A Locally Deployable Nanofiber Patch for Sequential Drug Delivery in Treatment of Primary and Advanced Orthotopic Hepatomas ∥

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Jiannan Li,†,‡, Weiguo Xu,†, Di Li,†, Tongjun Liu,‡ Yu Shrike Zhang,*,§ Jianxun Ding,†,* and Xuesi Chen†,* †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, 5625 Renmin Street, Changchun 130022, P. R. China ‡

Department of General Surgery, The Second Hospital of Jilin University, 218 Ziqiang Street,

Changchun 130041, P. R. China §

Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital,

Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA

ACS Paragon Plus Environment

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KEYWORDS: emulsion-electrospun polymer patch, core−sheath nanofiber, local drug codelivery, hepatoma, synergistic chemotherapy

ABSTRACT: With unsatisfactory effects of systemic chemotherapy for treatment of unresectable or advanced hepatoma, local and sustained delivery of chemotherapeutic agents is becoming a promising solution. The in situ administrated platforms increase the drug concentrations in tumor regions, decrease the side effects to organs, prevent the damages of vascular endothelium, and reduce the frequency of drug administration. The prevalent strategy based on minimally invasive transarterial chemoembolization (TACE) oftentimes induces upper gastrointestinal hemorrhage, liver failure, and liver abscess. In addition, integrating various antitumor drugs in one platform, especially the drugs with different hydrophilic/hydrophobic properties, and achieving sustained and/or sequential release profiles to synergistically inhibit cancer development, remains challenging. In this study, a local drug delivery system made of an emulsion electrospun polymer patch was developed, which contained hydrophobic 10hydroxycamptothecin (HCPT) and hydrophilic tea polyphenols (TP) in the shell and core of the nanofiber, respectively. Due to this core−sheath structure, HCPT and TP exhibited sustained and sequential releases first with HCPT followed by TP. HCPT was used to suppress the proliferation and malignant transformation of hepatoma, while TP was aimed to decrease the levels of oxygen free radicals and further prevent the invasion and metastasis of the tumor cells. Our study presented the potential superiority of this class of core−sheath structured nanofiber membranes in localized treatment of both primary and advanced orthotopic hepatomas.


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Liver is one of the most important internal organs performing critical functions, such as storing glycogen and producing bile to help with digestion, among others. Hepatoma, including primary and metastatic hepatomas, is the 3rd most deadly cancer and a major clinical problem.1 The poor prognosis and high recurrence rate following resection are two prevalent problems. Because most of the patients are diagnosed at the intermediate to advanced stages of hepatoma, the traditional treatment methods, such as resection, percutaneous ablation, and liver transplantation, are not suitable.2 For patients with advanced hepatoma, transarterial chemoembolization (TACE) is currently considered to be the fist-line therapy in the clinic.2 However, the benefit of TACE for patients is typically small and it is oftentimes inclined to lead to life-threatening complications, such as the upper gastrointestinal hemorrhage, liver failure, and liver abscess due to their inability to degrade.3,4 Systemic chemotherapy of intravenously or arterially injecting targeted antitumor drugs (e.g., sorafenib) is another clinical method for unresectable hepatoma. However, the therapeutic effect and the increased survival time of systemic chemotherapy towards hepatoma are not satisfactory.3 In addition, long-term systemic chemotherapy can lead to undesired side effects to many other healthy organs, as well as the damages to the endothelium of blood vessels. It is reported that the damages of the vascular endothelium can not only restrict the chemotherapeutic effects but also enhance the invasion and metastasis of tumor cells.4,5 To overcome the disadvantages of the abovementioned treatments for unresectable hepatoma, local chemotherapy that utilizes antitumor agent-loaded electrospun nanofibers has attracted increasing attention.6 The nanofibers possess the abilities of high drug encapsulation efficacy, better stability, sustained release of drugs, high surface contact area, and degradability.7,8 As a result, local and sustained delivery of chemotherapy drugs can increase the local drug concentrations in the tumor area, control and delay the drug release at cytotoxic concentrations


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and longer periods, decrease the injuries to other organs, prevent the damages to vascular endothelium, and reduce the frequency of drug administration. Although the implantation of the electrospun membrane for hepatoma treatment needs open surgery, this limitation can be minimized by the utilization of interventional technologies such as catheters for minimally invasive delivery. Therefore, local chemotherapy that relies on antitumor agent-loaded electrospun membranes might be promising for the treatment of unresectable hepatoma. Cancer is considered as a genetic disease, in which the proliferation of tumor cells is not tightly regulated anymore. During the development of cancer, the tumor cells rapidly divide and proliferate, followed by the malignant transformation.9 When the malignant transformation happens, the tumor cells become more invasive and cancer metastasis can occur.10 In addition, the mitochondria dysfunction, which can induce the generation of large amounts of reactive oxygen species (ROS), becomes more and more pronounced during the malignant transformation process.11 It is reported that high level of ROS is inclined to promote the invasion and metastasis of cancer.11 As a result, an effective localized cancer treatment might be divided into two steps. First, the proliferation of tumor cells should be well-suppressed to decrease the malignant transformation. Second, the formation of ROS should be inhibited to further prevent the invasion and metastasis of tumor cells. In particular, 10-hydroxycamptothecin (HCPT) is a type of commonly used topoisomerase-1 inhibitor and has widely shown promising antitumor properties.12,13 Generally, HCPT can inhibit the DNA replication and efficiently prevent the proliferation of cancer cells.14 The formation of ROS on the other hand, can be obviously decreased by phenolic derivatives, especially the tea polyphenols (TP).15 TP extracted from green tea has been studied in many cancer prevention researches and has shown significant ROS inhibition properties.15,16 Furthermore, HCPT and TP


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also induce the mitochondria-related apoptosis in cancer cells through increasing caspase-3 expression.17−19 Therefore, HCPT and TP were chosen to synergistically prevent the hepatoma progression in our study. Considering the fact that the hydrophilic/hydrophobic properties of HCPT and TP are quite different, integrating these two drugs in one platform is difficult. In addition, achieving sequential releases first with HCPT followed by TP in a sustained manner is challenging using conventional drug delivery strategies such as TACE. In contrast, the structure of the nanofiber can be facilely modulated through emulsion electrospinning, which separates the oil and aqueous phases in the sheath and core, respectively. Based on this core−sheath structure, agents with different hydrophilic/hydrophobic properties can be encapsulated within a single platform.20 As a result, the hydrophobic and hydrophilic drugs may both exhibit sustained release behaviors of hydrophilic drug decelerating and keeping a similar rate with the hydrophobic drug for improved synergistic effects against cancer. Herein, an emulsion electrospun polymer patch composed of core−sheath nanofiber was prepared. HCPT and TP were encapsulated within the shell and core of the nanofibers, respectively, to treat both primary and advanced orthotopic hepatomas (Scheme 1). Through emulsion electrospinning, methoxy poly(ethylene glycol)-block-poly(lactide-co-glycolide) (mPEG-b-PLGA) and dextran emulsion with HCPT in the oil phase and TP in the aqueous phase was developed. After patching the membrane to the orthotopic hepatoma, these two drugs were released sequentially and sustainably to exhibit antiproliferative and antioxidant properties, respectively. With our rationally designed structure, the membrane achieved local delivery of HCPT and TP to synergistically inhibit the progression of primary and advanced orthotopic hepatomas. Unlike other similar studies that utilized local chemotherapy for the treatment of orthotopic hepatoma,6,21 the dual-drug loaded membrane used in our study successfully


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decreased the malignant transformation first and then prevented the invasion and metastasis of tumor cells by integrating HCPT and TP in a single platform achieving their sequential release.


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Scheme 1. Schematic illustration of preparation of HCPT and TP co-loaded emulsionelectrospun membrane (EEPM/TP+HCPT), construction of POH and AOH models, and synergy mechanism of EEPM/TP+HCPT against hepatoma. RESULTS For simplicity, EEPM, EEPM/HCPT, EEPM/TP, and EEPM/TP+HCPT represented the blank emulsion-electrospun membrane, and the membranes loaded with HCPT, TP, and TP+HCPT, respectively. Characterizations of Emulsion-Electrospun Membranes. EEPM/TP+HCPT, EEPM/HCPT, EEPM/TP, and EEPM were successfully produced by emulsion electrospinning. The core−sheath structure of nanofiber was developed and assessed by transmission electron microscope (TEM; JEOL; Tokyo, Japan) and confocal laser scanning microscope (CLSM; Carl Zeiss, LSM 780; Jena, Germany). The presence of the core−sheath structure of the nanofiber was apparent, and the boundary between the two layers was sharp (Figure 1A). The average diameter of the inner core was 46.6 ± 28.6 nm. Furthermore, the inner core of the nanofiber was incorporated with rhodamine B (RhB) and analyzed through CLSM. The inner core emitted distinct red fluorescence, while no red fluorescence was present in the outer sheath (Figure 1B). The surface morphologies of EEPM/TP+HCPT, EEPM/HCPT, EEPM/TP, and EEPM were evaluated by SEM (Figure 1C). The nanofibers were smooth and homogenous in all groups, and no drug crystal was observed on the surface. The water contact angles of these four membranes were all above 100°, and there was no statistical difference (Figure 1C). The frequency distributions of nanofiber diameters and pore sizes were analyzed and both of them showed Gaussian distributions (Figure S2, Supporting Information). The average nanofiber diameters of EEPM/TP+HCPT, EEPM/HCPT, EEPM/TP, and EEPM were 139.0 ± 25.6, 138.1 ± 24.7, 146.5


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± 23.3, and 135.2 ± 28.6 nm, respectively, while the average pore sizes were 196.2 ± 35.2, 194.0 ± 42.1, 168.5 ± 35.0, and 179.4 ± 49.3 nm, respectively.

Figure 1. Properties of the emulsion-electrospun membranes. (A) TEM and (B) CLSM images of emulsion electrospun nanofiber. (C) SEM and water contact angles analyses. Scale bars = 1 µm. (D, E, F, and G) Mechanical properties of different membranes, including (D) stress-strain


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curve, (E) elongation at break, (F) tensile strength, and (G) Young’s modulus. Data are presented as mean ± standard deviation (SD; n = 3). The mechanical properties of all the membranes are shown in Figure 1D–G. There were no statistical differences among the four types of membranes in mechanical characteristics. In Vitro Degradation. Figure 2A shows the in vitro degradation of EEPM in phosphatebuffered saline (PBS) in the presence and absence of elastase. The degradation rate of EEPM was faster in the elastase solution compared with that in PBS (P < 0.001). After degradation for 30 days in PBS with and without elastase, the weight losses of EEPMs were 15.7 ± 2.0 wt.% and 76.3 ± 2.1 wt.%, respectively. In Vitro Drug Release. The drug loading contents (DLCs) of HCPT and TP were 5.4% and 5.3%, respectively. The drug loading efficiencies (DLEs) of HCPT and TP were 90.5% and 89.1%, respectively. The accumulative HCPT release profiles for EEPM/HCPT and EEPM/TP+HCPT, and the TP release profiles for EEPM/TP and EEPM/TP+HCPT, in PBS, are shown in Figure 2B and 2C, respectively. No statistical difference of HCPT release profiles was observed between the EEPM/HCPT and EEPM/TP+HCPT groups. During the initial 4 days, burst releases of 62.6% ± 3.3% and 61.5% ± 2.5% were found in the EEPM/HCPT and EEPM/TP+HCPT groups, respectively. Sustained fast releases of HCPT were detected for the next 6 days, followed by slow releases for 9 more days. After 19 days, 87.9% ± 4.1% and 91.3% ± 2.9% of HCPT were released in the EEPM/HCPT and EEPM/TP+HCPT groups, respectively. In comparison, the TP release profiles were different from those of HCPT. Only small amounts of TP were released in the first 3 days (16.3% ± 4.0% in the EEPM/TP group and 20.4% ± 2.2% in the EEPM/TP+HCPT group), followed by constant fast releases for the next 7 days. After 19


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days, the released TP amounts were 89.2% ± 2.0% and 88.1% ± 2.3% in the EEPM/TP and EEPM/TP+HCPT groups, respectively.

Figure 2. Degradation and drug release behaviors of the membranes. (A) Degradation properties of EEPMs. (B, D) HCPT release behaviors of EEPM/HCPT and EEPM/TP+HCPT in PBS (B) and elastase solution (D). (C, E) TP release behaviors of EEPM/TP and EEPM/TP+HCPT in PBS (C) and elastase solution (E). (F) In vivo EEPM degradation profile. (G) Morphologies and representative SEM images of degraded EEPMs at pre-set intervals. Red arrows indicated the degraded membranes. Scale bars are 1 cm and 2 µm for morphologies and SEM images of


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degraded EEPMs, respectively. Data are presented as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001). Figure 2D and 2E shows HCPT and TP release profiles in the elastase solution. For the first 3 days, 67.0% ± 2.4% and 65.1% ± 3.5% of the total amounts of HCPT were observed to release in the EEPM/HCPT and EEPM/TP+HCPT groups, respectively. Constant fast releases were found for the next 3 days, followed by very slow release profiles. After 10 days, the released HCPT were 92.2% ± 0.5% and 91.2% ± 1.1% in the EEPM/HCPT and EEPM/TP+HCPT groups, respectively. Compared with the HCPT release profiles, those of TP were slower. For the first 3 days, 43.8% ± 4.1% and 45.7% ± 5.2% of TP were released in the EEPM/TP and EEPM/TP+HCPT groups, respectively. Constant fast releases were found for the next 5 days followed by slow releases. After 13 days, the released TP in the EEPM/TP and EEPM/TP+HCPT groups were 91.1% ± 0.3% and 90.9% ± 1.0%, respectively. Figure S3 and Table S1 show the cumulative drug release from the 1.0 × 1.0 cm2-sized EEPM/TP+HCPT membranes in elastase solution over time. Importantly, the sequential release of HCPT and TP was confirmed in Figure S4 (Supporting Information). For the first 3 days, an abrupt release of HCPT from EEPM/TP+HCPT was detected with a k (the release rate coefficient) of 0.24. However, the release profile of TP (k = 0.17) was slower than that of HCPT at the same time. During the next 3 days, the release rate of TP (k = 0.12) was much faster than that of HCPT (k = 0.08).


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Figure 3. Treatment of POH in mice. (A) Experimental schedule. (B) Photographs of liver tumors in the 2nd week post-treatment in different groups. (C) Tumor volumes, (D) tumor weight, and (E) body weight changes. (F) Organ coefficients. White arrows indicated the tumors. Scale bar = 1 cm. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001). In EEPM/TP+HCPT, EEPM/HCPT, and EEPM/TP groups, n = 6. In EEPM and control groups, n = 5. In Vivo Degradation. The in vivo degradation study of EEPM was performed for 7 weeks. Figure 2F shows the remaining weight of the membrane at different time intervals. The remaining weight of EEPM increased in the 1st week, and then decreased. In the 1st, 3rd, 5th, and 7th week, the remaining weights of EEPM were 111.2% ± 3.8%, 79.8% ± 5.5%, 54.6% ± 2.2%, and 42.1% ± 5.5%, respectively. The morphologies of degraded EEPM at each time interval were photographed and analyzed by SEM (Figure 2G). EEPM became smaller in size as it degraded. In the 1st week post-implantation in vivo, some of the nanofibers became obscure and fused, but their basic structures were still retained. In the 3rd week, only a small amount of nanofibers remained their basic structures. In the 5th and 7th week, there were not any visible nanofiber structures, but instead were the fused mixtures without concrete morphologies. Effects of EEPM/TP+HCPT against Hepatoma. The primary orthotopic hepatoma (POH) model was successfully developed to investigate the effects of EEPM/TP+HCPT against hepatoma. Due to the surgical injuries of two laparotomies and the tumor cachexia, one animal in the EEPM group and another animal in the control group died during the observation process. After the euthanasia of mice, the proper integration of the membranes and the liver tissues was examined first. The results confirmed that none of the membranes dislodged from the sites of action after 14 days of treatment.


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Figure 4. Treatment of AOH in mice. (A) Experimental schedule. (B) Photographs of liver tumors in the 2nd week post-treatment in different groups. (C) Tumor volume, (D) tumor weight,


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and (E) body weight changes. (F) Organ coefficients, (G) ascites volume, and (H) abdominal circumference changes. (I) Cell number of ascites in mice. (J) Cell viability of ascites. White arrows indicate the tumors. Scale bar = 1 cm. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001). In EEPM/TP+HCPT, EEPM/HCPT, and EEPM/TP groups, n = 5. In EEPM and control groups, n = 4. Figure 3A shows procedure of the in vivo experiment, including orthotopic liver injection of H22 cells, electrospun patch implantation, and tissue analyses. Morphological assessment of the liver showed that most of the livers in the drug-loaded groups (6/6 in the EEPM/TP+HCPT group, 5/6 in the EEPM/HCPT group, and 5/6 in the EEPM/TP group) remained normal appearance. However, in the EEPM and control groups, the tumors were large, protruded from the surface of the liver, and occupied large sections of the livers (Figure 3B). In comparison with control group, the tumor volumes were relatively smaller in drug-loaded groups (Figure 3C). Particularly, the tumor volume prominently decreased in the EEPM/TP+HCPT group. Consistent with the tumor volume, the EEPM/TP+HCPT group also presented the least tumor weight (Figure 3D). The changes of the body weight of mice are shown in Figure 3E. After each surgical procedure, the body weight of mice decreased for the initial 3 or 4 days, and then increased. After treatment for 2 weeks, there was statistical difference in the body weight of mice between EEPM/TP+HCPT and control groups (P < 0.05). The organ coefficients that can represent the basic organ functions are shown in Figure 3F, and no statistical difference was observed in all the groups.


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Figure 5. Biochemical changes in both animal models. (A, B) Biochemical analyses of ALT, AST, AFP, CK-MB, and BUN in the (A) POH and (B) AOH models. Data are presented as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001). The efficacy of EEPM/TP+HCPT against the advanced orthotopic hepatoma (AOH) was also investigated. In the 3rd week after in situ liver injection of H22 cells, there were five mice left in the EEPM/TP+HCPT, EEPM/HCPT, and EEPM/TP groups, and four mice left in the EEPM and control groups, due to the two laparotomies surgical injuries and the tumor cachexia. Figure 4A shows the experimental procedure, including orthotopic liver injection of H22 cells, electrospun patch implantation, intraperitoneal injection of H22 cells, and tissue analyses. Morphological observation of the livers indicated that nearly all the tumors in the EEPM and control groups protruded from surface of the livers and occupied a large section of the livers (Figure 4B). The EEPM/TP+HCPT group showed the least tumor volume among all the groups (Figure 4C). Consistent with the tumor volume, the tumor weight was also the least in the EEPM/TP+HCPT group (Figure 4D). The body weights of mice with AOH are shown in Figure 4E. In all groups, the body weights of mice decreased after each surgical procedure, and then increased. In the 2nd week after electrospun patch implantation, there was statistical difference in the body weights of mice between the EEPM/TP+HCPT and control groups (P < 0.05). As for the organ coefficients, there was also no obvious difference among all groups (Figure 4F). The ascites volume and abdominal circumference changes are provided in Figure 4G and 4H. In comparison with the control group, the EEPM/TP+HCPT group presented higher ascites inhibition efficacy (P < 0.01). The abdominal circumferences increased among all groups, while the EEPM/TP+HCPT group had the slowest rate of increase. The number of tumor cells in ascites and the cell viability were also


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examined using an automatic cell counter. Figure 4I shows that the cell number was the least in the EEPM/TP+HCPT group. In addition, the EEPM/TP+HCPT group also decreased the cell viability significantly (Figure 4J). To assess the general condition of the liver, alanine transaminase (ALT), aspartate aminotransferase (AST), and a-fetoprotein (AFP) were analyzed in both models. In addition, creatine kinase MB isoenzyme (CK-MB), and blood urea nitrogen (BUN) levels were also examined to evaluate the heart and kidney conditions. Figure 5A shows the blood biochemical values in the POH model, and it indicated that the ALT, AST, and AFP levels increased in tumor-bearing mice compared with those in healthy mice. In comparison to the control group, the EEPM/TP+HCPT group had the lowest levels of ALT, AST, and AFP. However, there was no clear difference in CK-MB and BUN levels among all groups. In the AOH model, these blood biomarker levels, including ALT, AST, AFP, CK-MB, and BUN, were similar with those of the POH model (Figure 5B). In comparison with the control group, the EEPM/TP+HCPT group more effectively decreased ALT, AST, and AFP levels. Furthermore, no statistical difference was observed in CK-MB and BUN values among all groups.


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Figure 6. Histological analyses of antitumor efficacies. Data in A−D originated from the POH model, and in E−H represent the AOH model. (A, E) H&E staining of liver tumors in the left liver lobe. Red boundaries indicate the original tumor areas in the POH mice. a−e represent the control, EEPM/TP+HCPT, EEPM/HCPT, EEPM/TP, and EEPM groups, respectively. (B, F) H&E staining of other liver lobes. Red boundaries indicate the original tumor areas in the AOH mice. (C, G) Cross-sectional areas of tumor in the left liver lobes. (D, H) Cross-secctional areas of metastatic tumors in different groups. Scale bars are 5 mm for (A) and (E), and 1 mm for (B) and (F). Data are presented as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001). To comprehensively and clearly evaluate the antitumor efficacy of EEPM/TP+HCPT against POH and AOH, hematoxylin and eosin (H&E) staining of liver sections in each liver lobe was further performed. Figure 6A and 6E shows the histological analyses of the tumor-loaded liver lobes in different groups of the POH and AOH models, respectively. In both models, the margins between the tumor tissues and normal liver tissues were sharp. The tumor cross-sectional area in each group was analyzed by Adobe Photoshop CS6 (Adobe; USA). In comparison with the control group, the drug-loaded groups better decreased the cross-sectional tumor areas in the POH model (Figure 6C). The smallest cross-sectional tumor area was found in the EEPM/TP+HCPT group. In the AOH model, according to the tumor cross-sectional area analyses, the EEPM/TP+HCPT group also exhibited the best antitumor property (Figure 6G). H&E staining of other liver lobes was also performed to evaluate the tumor invasion. Tumor invasiveness occurred in the left lateral and right liver lobes in the control group, and the left lateral liver lobe in EEPM/TP and EEPM groups (Figure 6B). Among these three groups, the EEPM/TP group had the smallest cross-sectional area (Figure 6D). In the AOH model, the left lateral and right liver lobes in the control group, and the left lateral liver lobe in EEPM group


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presented invasive hepatoma (Figure 6F). There was statistical difference in the cross-sectional area of invasive tumor between control and EEPM groups (Figure 6H, P < 0.001). H&E staining of other major organs was also performed to evaluate the tumor metastasis and tissue injuries (Figure S5, Supporting Information). No evidence of tumor metastasis was observed in these organs in both models. In addition, tissue injuries were not obvious.

Figure 7. Immunfluorescent and Western blot analyses of tumor tissues in different groups in POH model. (A) Immunofluorescence analyses of expression levels of PCNA, ROMO1, and caspase-3 in tumor tissues. (B) Western blot analyses of expressed protein levels of PCNA, ROMO1, and caspase-3 in the tumor tissues. (C) Relative optical densities of tumor sections from PCNA, ROMO1, and caspase-3. (D) Levels of protein expression using semiquantitative analyses. Actin was used as a control. Scale bar = 50 µm. Data are presented as mean ± SD (for C, n = 3; for D, n = 5; *P < 0.05, **P < 0.01, ***P < 0.001).


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Immunofluorescence and Western Blot Analyses. Immunofluorescence staining of proliferating cell nuclear antigen (PCNA), reactive oxygen species modulator 1 (ROMO1), and caspase-3 were performed to further evaluate the tumor inhibition mechanisms of EEPM/TP+HCPT in both the POH model (Figure 7A) and the AOH model (Figure 8A). For PCNA and ROMO1 analyses, the weakest and strongest fluorescence intensities were found in the EEPM/TP+HCPT and control groups for both models, respectively. For caspase-3 evaluation, the EEPM/TP+HCPT group presented the strongest fluorescence intensities, compared with other groups. The results were further confirmed by semi-quantitative analyses. In the POH model, the least amounts of PCNA and the largest amounts of caspase-3 were found in the EEPM/TP+HCPT group (Figure 7C). In addition, the EEPM/TP+HCPT group inhibited the expression of ROMO1 greatly, but no statistical difference was observed between the EEPM/TP+HCPT and EEPM/TP groups (Figure 7C, P > 0.05). In the AOH model, the expression of PCNA was best inhibited in the EEPM/TP+HCPT group. The expression of ROMO1 was also best inhibited in the EEPM/TP+HCPT group, and the statistical difference was significant between the EEPM/TP+HCPT and EEPM/TP groups (P < 0.05) (Figure 8C). Furthermore, the largest amounts of caspase-3 were found in the same group (Figure 8C). The protein levels of PCNA, ROMO1, and caspase-3 in tumor tissues in the POH model (Figure 7B) and the AOH model (Figure 8B) were observed by Western blot. In the POH model, the expression of PCNA was best inhibited in the EEPM/TP+HCPT group. The protein levels of ROMO1 were significantly lower in the EEPM/TP+HCPT, EEPM/HCPT, and EEPM/TP groups than those of the control and EEPM groups (Figure 7D). In addition, the protein level of caspase3 was higher in the EEPM/TP+HCPT group than those of any other groups (Figure 7D). In the AOH model, the EEPM/TP+HCPT, EEPM/HCPT, and EEPM/TP groups all presented lower


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PCNA expression levels than those of the control and EEPM groups (Figure 8D). As for PCNA expression, no significant difference was observed among all the drug-loaded groups. The protein level of ROMO1 was the lowest in the EEPM/TP+HCPT group (Figure 8D). As for the level of caspase-3, the EEPM/TP+HCPT group showed the most amounts (Figure 8D).

Figure 8. Immunfluorescent and Western blot analyses of tumor tissues in different groups in AOH model. (A) Immunofluorescence analyses of expression levels of PCNA, ROMO1, and caspase-3 in tumor tissues. (B) Western blot analyses of expressed protein levels of PCNA, ROMO1, and caspase-3 in the tumor tissues. (C) Relative optical densities of tumor sections from PCNA, ROMO1, and caspase-3. (D) Levels of protein expression using semiquantitative analyses. Actin was used as a control. Scale bar = 50 µm. Data are presented as mean ± SD (for C, n = 3; for D, n = 5; *P < 0.05, **P < 0.01, ***P < 0.001).


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DISCUSSION In this study, core−sheath structured nanofibers loaded with hydrophobic HCPT in the sheath and hydrophilic TP in the core were prepared for in situ chemotherapy of hepatomas. These two drugs were integrated in one drug delivery system through emulsion electrospinning. HCPT was chosen for its topoisomerase-1 inhibition property, which can inhibit DNA replication, RNA transcription, and prevent proliferation of tumor cells.22,23 TP was used for its antioxidant activity, which can scavenge oxygen free radicals and inhibit tumor invasion and metastasis.15 As the nanofiber degraded, HCPT that was located in the sheath of fiber could be released first, followed by TP. The sequential and sustained release of HCPT and TP from the emulsion electrospun nanofiber was aimed to prevent the proliferation of tumor cells, decrease the level of ROS, and synergistically achieve a higher efficacy of tumor apoptosis and hepatoma inhibition. Orthotopic hepatoma models were performed to better mimic the original hepatoma growth and development. Besides the POH model, mice were also intraperitoneally injected with H22 cells to simulate the advanced hepatoma, intending to evaluate the treatment efficacy of EEPM/TP+HCPT against both POH and AOH. The core−sheath structure of the nanofiber was confirmed by TEM and CLSM. Under CLSM observation, the red fluorescence was almost entirely located in the core, which was labeled with RhB. Representative SEM images indicated that the emulsion electrospun nanofiber was fabricated successfully. The surface of the nanofibers was smooth, and the diameters and pore sizes were homogeneous. Such an observation was possibly attributed to the suitable electrospun parameters and good solubility of drugs, including HCPT in chloroform (CHCl3) and TP in water. Mechanical tests and water contact angle analyses showed that incorporation of HCPT and TP did not change the mechanical properties or hydrophilicity of the membranes. This was


24

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because that only small amounts of drugs (6.0 wt.% HCPT and/or TP with respect to mPEG-bPLGA) were applied in this study. In vitro and in vivo degradation studies were performed to analyze the biocompatibility of different membranes. For in vitro degradation, the weight loss had a steep decline rate in the elastase solution group, where the average rate was about 80% after incubation for 30 days. In clear contrast, the weight loss rate was less than 20% after incubation for 30 days in the PBS group. This was due to the properties of elastase solution, which decomposed the polymers quickly. The in vivo degradation study indicated the general weight changes and degradation characteristics of EEPM. During this process, many cells, proteins, and other biomacromolecules may be adsorbed onto the surface of the implants. However, in our study, the implanted membranes were carefully rinsed and cleared to remove the adsorbed proteins and cells to ensure the veracity of the in vivo degradation results. The weight of EEPM increased in the 1st week post-percutaneous implantation. This was possibly because that EEPM acted as the foreign materials, which induced foreign body responses and increased the infiltration of inflammatory cells and proteins into the nanofibers.24 This was consistent with Wang’s study, in which the weight of electrospun membranes also increased in the 1st week post-degradation in vivo.24 Even though the degradation process reduced the weight of EEPM, the cell infiltration might have also induced the weight increase. At the early stage of infiltration, the weight increase overpassed the weight decrease, leading to the weight increase of EEPM in the 1st week. Consistent with the degradation process of EEPM, the membranes became smaller and obscure. In addition, SEM evaluations also verified the increased degradation of EEPM. The sequential and sustained releases of HCPT and TP from the membranes are potentially significant for pharmaceutical treatment of hepatoma. Consistent with the degradation properties


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of the membranes in PBS with and without elastase, both drugs presented fast release profiles in elastase solution and relative slow release profiles in PBS. In both types of drenching solution, especially in the presence of elastase, the burst releases of the drugs were obvious. This observation could be attributed to the distribution of drugs near the surface of the nanofibers, large surface area of the nanofiber mats, and quick degradation of the membranes. In the elastase solution, the burst releases of HCPT and TP were obvious for the first 3 days, and the total release times of HCPT and TP were 10 and 13 days, respectively. The rapid releases of the two drugs could increase the concentrations of drugs at the tumor site and inhibit tumor growth.6 Importantly, the drug release rate results confirmed that HCPT and TP exhibited a sequential release behavior with HCPT released quickly at the beginning and TP possessing a sustained release profile. In addition, the burst release of TP was not significant compared to that of HCPT and the TP release was slower than that of HCPT, because TP was mainly located in the core of the nanofiber, whereas HCPT were in the sheaths. The sequential releases of these two drugs aimed to synergistically prevent the proliferation, invasiveness, and metastasis of hepatoma. Because HCPT and TP possessed good dissolution in oil and aqueous phases, respectively, the sustained release profiles of both drugs could provide long-term effects to suppress the hepatoma progression.6 Controllable release of drugs from electrospun fibers has already been studied. In one study, the authors developed mitomycin-C (MMC)-loaded poly(L-lactic acid) (PLLA) micro-sol electrospun nanofibrous membranes.25 The water-soluble MMC located in the inner core of the fibers exhibited a sustained release behavior for approximately 1 month in PBS. Zhao et al. reported a pH-responsive doxorubicin (DOX) release system made up of PLLA nanofiber encapsulating mesoporous silica nanoparticles (MSNs) with a CaCO3 cap.26 DOX could be


26

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released fast in the acidic environment and the total release time was more than 30 days. In comparison, our study simultaneously incorporated two classes of drugs in one electrospun platform and well-controlled the release profiles of them. Sequential release of drugs from electrospun fibrous scaffolds was also achieved previously. Zhao et al. developed polydopamine (PDA)-coated MSNs encapsulated in PLLA microfibers.27 DOX and ibuprofen (IBU) were incorporated in MSNs and electrospun microfibers, respectively. IBU showed a shorter-term release for 30 days while DOX demonstrated a longer-term release for 90 days. Different from Zhao’s study, our study allows easy control over the sequential release of drugs with different hydrophobic/hydrophilic properties due to the core−sheath structure of the fibers. To further examine the antitumor effect of EEPM/TP+HCPT in vivo, both POH and AOH models were constructed. Compared with the traditional subcutaneous tumor models, orthotopic tumor models are considered more clinically meaningful and theoretically predicable in efficacy testing of drugs.28 It is anticipated that, directly implanting tumor cells within the liver tissues can simulate the original conditions of tumor growth, invasiveness, metastasis, and recurrence. In addition, injecting tumor cells intraperitoneally was used to mimic the advanced hepatoma and to detect the treatment efficacy of EEPM/TP+HCPT. Malignant ascites is a common symptom of highly advanced cancer and may lead to further metastasis of tumor cells.29 In the AOH model used in our study, the developed malignant ascites and in vivo hepatoma were believed to sufficiently mimic the highly malignant liver cancer and demonstrate the treatment efficacy of our membranes. To better photograph the liver tumors and measure the tumor volumes, the membranes were carefully removed before the histology analyses. As a result, no histology result showing the interface of tissue and scaffold was obtained. However, the integrations of the scaffolds to liver


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tissues were examined after the euthanasia of mice confirming no dislodgement of the membranes from the sites of application. Due to the controlled releases of both HCPT and TP and the chemotherapeutic effect of these two drugs, EEPM/TP+HCPT presented satisfactory anti-hepatoma treatment efficacy in our study. After treatments, EEPM/TP+HCPT greatly decreased the tumor volume and tumor weight compared with the EEPM/TP and EEPM/HCPT groups. In the AOH model, EEPM/TP+HCPT also greatly reduced the ascites volume and abdominal circumference in comparison to the control group. Furthermore, the number of tumor cells was the least and the cell viability of ascites was the lowest in the EEPM/TP+HCPT group. In neither animal model, EEPM showed any tumor inhibition effect as no drug was loaded in this membrane.

Histopathological

analyses

also

confirmed

the

treatment

efficacy

of

EEPM/TP+HCPT, with less tumor cross-sectional area and no tumor invasiveness in other liver lobes. Also, the EEPM/TP+HCPT group showed smaller tumor cross-sectional area when compared with those of the EEPM/TP and EEPM/HCPT groups. Even though the EEPM/TP and EEPM/HCPT groups inhibited the tumor growth compared with control group, the EEPM/TP+HCPT groups presented better tumor inhibition efficiency. This might be attributed to the fact that HCPT prevented the proliferation of cancer cells, and TP suppressed the ROS level that was associated with the invasiveness and metastasis of hepatoma. The synergistic effect of these two drugs better prevented the progression of hepatoma than that of the single drug treatments. In addition, the general liver function observations, including ALT, AST, and AFP analyses, reflected the improved state of the livers in the EEPM/TP+HCPT group. As a new treatment method for orthotopic hepatoma, the safety of the membranes should be concerned. The body weights of mice varied between the EEPM/TP+HCPT and control groups, which indicated that the dual drug-treated mice were in better body conditions. This is because of


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the therapeutic effect of TP and HCPT and the less-toxic properties of the membrane. There were not any statistical differences in organ coefficients among different groups. In addition, the histopathology of the heart, spleen, lung, and kidney did not indicate obvious organ injuries. Heart and kidney functions were also analyzed by testing CK-MB and BUN levels in the serum. Even though in cases some slight cell degeneration are found due to the acute toxicity of the burst release phenomenon of drugs, the structure of liver sinusoid and cord can usually recover quickly.6 In our study however, no obvious side effects were observed in liver tissues 14 days post-treatment. In addition, no side effects were found in normal organs as well. As a result, EEPM/TP+HCPT was safe in the treatment of orthotopic hepatoma in this study. Liu et al. also demonstrated the safety of chemotherapy drug-loaded electrospun nanofibers for the treatment of orthotopic hepatoma.6 In their study, the body weights of mice treated with or without doxorubicin-loaded electrospun membranes all recovered rapidly and increased steadily during the whole experiment process. In addition, other organs were normal post-treatment. These results indicated that electrospun membranes incorporated with chemotherapy drugs were safe for hepatoma treatment. To further reveal the mechanism of HCPT and TP in the treatment of hepatoma, immunofluorescence and Western blot analyses of the levels of PCNA, ROMO1, and caspase-3 were performed. As a nuclear protein, PCNA is upregulated with DNA repair, and is related with tumor cell proliferation.22,30 ROMO1 is a class of protein, which is related with the high level of ROS.31,32 ROS, generated by oxygen free radicals, is known to be associated with the invasive and proliferative abilities of tumor cells.33 It can also lead to DNA damage and mutations.34 Most of the antitumor drugs utilize the general apoptotic mechanism to trigger tumor cell death, and caspase-3 is the fundamental regulator of apoptosis.35 As a result, the expressions of PCNA and


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ROMO1 were evaluated to analyze the treatment mechanism of EEPM/TP+HCPT against hepatoma. Casepse-3 levels were assessed to study the apoptotic effect of cancer cells by EEPM/TP+HCPT. The down-regulated PCNA and ROMO1 in the EEPM/TP+HCPT, EEPM/HCPT, and EEPM/TP groups indicated that HCPT and TP released successfully from membranes and played their specific effect on hepatoma treatment. In comparison to the EEPM/HCPT and EEPM/TP groups, the EEPM/TP+HCPT groups better decreased the expression of PCNA and ROMO1. While HCPT and TP mainly performs anti-proliferation and antioxidant functions in cancer treatment, synergistic application of these two drugs apparently resulted in further decreased proliferation of cancer cells and lower levels of oxygen free radicals compared with single drug applications. In addition, the EEPM/HCPT and EEPM/TP groups upregulated caspase-3 compared with the control group, but the EEPM/TP+HCPT group showed the highest level of caspase-3 among all the groups. This may well explain the synergistic chemotherapeutic effect of HCPT and TP that the integration of these two drugs could better induce the apoptosis of tumor cells thus inhibiting the tumor growth. CONCLUSIONS In this study, a dual drug delivery system composed of HCPT and TP-loaded nanofibers was successfully developed through emulsion electrospinning. HCPT and TP were encapsulated in the sheath and core of the nanofibers, respectively. The membrane rationally integrated hydrophobic HCPT and hydrophilic TP in a single platform. Both primary and advanced hepatoma models were performed to analyze the treatment efficiency of the dual drugincorporated membrane. Due to the rationally designed core−sheath structure of the nanofibers, HCPT and TP were released sequentially in a sustained manner for up to 10 and 13 days, respectively. The dual drug-loaded membrane significantly suppressed the tumor proliferation


30

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and invasiveness in both animal models through the synergistic effect of HCPT and TP. All the results demonstrated the efficacy of this membrane in therapy of primary and advanced hepatomas. There are however, still some limitations associated with such membranes to be further applied for hepatoma treatment. The most important limitation lies in that open surgery is necessary to implant the membrane on the surface of the tumors, which may not be suited for certain patient populations. This disadvantage may be overcome through combination with interventional technologies using catheters or percutaneous needles for minimally invasive injection of the membranes for local deployment to reduce the negative effects of open surgery. This can possibly be achieved using shape-memory materials and will be explored in future iterations of the technological optimizations. In addition, whether obtained release profiles were ideal for the therapy cannot be confirmed. However, the tumor inhibition results indicated that the released HCPT and TP could efficiently prevent the hepatoma progression. In our future works, the membranes with varying drug release profiles will be further optimized. MATERIALS AND METHODS The materials, characterization of mPEG and mPEG-b-PLGA, preparation of emulsion electrospun nanofibers, characterization of membranes, degradation study, drug release study, and the immunofluorescence process can be found in the Supporting Information. Animal Study. BALB/c mice (female, 8−12 weeks old) were obtained from the Laboratory Animal Center of Jilin University and fed at Changchun Institute of Applied Chemistry, Chinese Academy of Science. Experimental Model. Two animal models were developed in this study: POH model, and AOH model. For the POH model, 0.1 mL of H22 cells (1.0 × 106 mL−1) were injected into left


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lobe of the liver in situ. For the AOH model, 0.2 mL of H22 cells (5.0 × 105 mL−1) were injected intraperitoneally in the 1st week after the injection of 0.1 mL of H22 cells (1.0 × 106 mL−1) in left lobe of the liver. The injection sites in liver tissues in both animal models were sealed by the thermocoagulation technique to avoid the outflow of H22 cells. The specific procedure of the establishment of orthotopic hepatoma model is shown in Figure S6 (Supporting Information). All the operations on the animals were in accordance with the guidelines of the United States National Institutes of Health (NIH) and approved by the Laboratory Animal Center of Jilin University. In Vivo Electrospun Membranes Treatment. For each animal model, mice were divided into five groups, i.e., control, EEPM/TP+HCPT, EEPM/HCPT, EEPM/TP, and EEPM, with six animals in each group. In the 1st week post-inoculation of H22 cells, 1.5 × 1.5 cm2 EEPM/TP+HCPT, EEPM/HCPT, EEPM/TP, and EEPM were directly pasted on the tumor surface after confirming the tumor formation through laparotomy. The membranes were sandwiched between the liver lobes to avoid the dislodgement from the tumor site. The control group did not receive any treatments. For the AOH model, H22 cells (200 µL, 5.0 × 105 mL−1) were injected intraperitoneally after suturing the abdomen. The body weight of each animal was monitored daily. For the AOH model, the abdominal circumference was also determined. In Vivo Antitumor Efficacy Evaluation. All the animals were euthanized in the 2nd week after implantation of membranes. Levels of ALT, AST, AFP, CK-MB, and BUN in the serum were analyzed by an enzyme-linked immunosorbent assay (ELISA). For the mice with AOH, abdominal ascites were collected, and each volume was measured. Then in each animal, 0.5 mL of abdominal ascites were obtained, centrifuged, rinsed, and diluted with 10.0 mL of PBS, and dyed with 0.4% Trypan blue solution. The tumor cell number and cell viability of the diluted


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ascites were analyzed by an automatic cell counter (NanoEnTek; Guro-gu, Korea), and then the cell concentrations of the original ascites were calculated. For the animals with their ascites volume of less than 0.5 mL, PBS was first used to dilute the original ascites to 0.5 mL. The heart, liver, spleen, lung, and kidney were isolated and weighed. The organ coefficients were obtained according to Eq. 1.

Organ Coefficient (%) =

W1 × 100% W2

(1)

In Eq. 1, W1 represents the organ mass and W2 represents the animal body weight. The livers were photographed first, and tumor sizes were measured. Then tumor volumes (V) were calculated by Eq. 2.

V (mm3 ) =

L × S2 2

(2)

In Eq. 2, L and S (mm) are the largest and smallest diameters of the tumor, respectively. Three samples in each group were selected, and the tumors in the livers were carefully dissected and weighed. Histological and Immunofluorescence Analyses. According to the basic anatomy, the liver tissues were separated into the left, left lateral, right, right lateral, and caudate lobes. These liver lobes were embedded, sectioned at 5.0-µm thick, and stained with H&E. Histological analyses were also performed for heart, spleen, lung, and kidneys tissues. The tumor tissues in the left hepatic lobe were analyzed and photographed under a microscope. The tumor cross-sectional area (cm2) was calculated by Adobe Photoshop CS6. Other hepatic lobes were also analyzed for metastatic tumors. For some other tumor-loaded liver sections, the immunofluorescence method was used to analyze the expression of PCNA, ROMO1, and caspase-3. Detailed processes of


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immunofluorescence

were

described

in

our

Page 34 of 42

previous

study.36

In

this

study,

the

immunofluorescence process can be found in the Supporting Information. Western Blot Analyses. The dissected tumors in each group were lysed with Total Protein Extraction Buffer (TransGen Biotech, Beijing P. R. China) for 60 min, and the lysates were centrifuged at 12,000 rpm for 10 min at 4 °C. The protein concentrations were determined and equal amounts (15.0 µg) of each sample were collected and separated. Then the gel was transferred to polyvinylidene difluoride (PVDF) membranes. The protein expressions were analysed by blocking the membranes in 5% skim milk for 1 h and incubating with PCNA, ROMO1, caspase-3, and β-actin antibodies. After incubation with secondary antibodies (1:5,000; Boster, P. R. China) for 40 min, the Western blot bands were analysed through an enhanced chemiluminescence (ECL) plus kit (Millipore, USA). Image J (NIH, Bethesda, Maryland, USA) software was used to quantify the densities of each band in a group, and then calculated their normalized levels. The protein densities of different groups were compared to assess the treatment efficacy of EEPM/TP+HCPT. Statistical Analyses. Data were presented as mean ± SD and analyzed using Student’s test when comparing 2 groups and one-way ANOVA when comparing more than 2 groups. *P < 0.05 indicated statistical significance, and **P < 0.01 and ***P < 0.001 indicated highly statistical significance.

ASSOCIATED CONTENT The authors declare no competing financial interest. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:


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Materials used in this study; characterization examination of mPEG and mPEG-b-PLGA; preparation of emulsion electrospun nanofibers; characterization test of membranes; in vitro and in vivo degradation; drug release study; immunofluorescence process; characterization of mPEG and mPEG-b-PLGA; distribution of diameters and pore sizes of different membranes; cumulative drug release from 1.0 × 1.0 cm2-sized EEPM/TP+HCPT in elastase solution over time; sequential release examination of HCPT and TP; H&E stainging of heart, spleen, lung, and kidney in both animal models; and establishment of orthotopic hepatoma animal model.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (J. Ding). *E-mail: [email protected] (Y. S. Zhang). *E-mail: [email protected] (X. Chen). ORCID Jiannan Li: 0000-0001-9744-7666 Weiguo Xu: 0000-0002-0146-8532 Di Li: 0000-0001-6783-4542 Tongjun Liu: 0000-0001-5712-4838 Yu Shrike Zhang: 0000-0002-0045-0808 Jianxun Ding: 0000-0002-5232-8863 Xuesi Chen: 0000-0003-3542-9256 Author Contributions ∥

J. Li, W. Xu, and D. Li contributed equally to this work.


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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Nos. 51673190, 51603204, 51673187, 51473165, and 51520105004) and the Science and Technology

Development

Program

of

Jilin

Province

(Nos.

20160204015SF

and

20160204018SF).

REFERENCES (1) Nelson, M. E.; Lahiri, S.; Chow, J. D. Y.; Byrne, F. L.; Hargett, S. R.; Breen, D. S.; Olzomer, E. M.; Wu, L. E.; Cooney, G. J.; Turner, N.; James, D. E.; Slack-Davis, J. K.; Lackner, C.; Caldwell, S. H.; Hoehn, K. L. Inhibition of Hepatic Lipogenesis Enhances Liver Tumorigenesis by Increasing Antioxidant Defence and Promoting Cell Survival. Nat. Commun. 2017, 8, 14689. (2) Huo, Y. R.; Eslick, G. D. Transcatheter Arterial Chemoembolization Plus Radiotherapy Compared with Chemoembolization Alone for Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis. JAMA Oncol. 2015, 1, 756–765. (3) Au, J. S.; Frenette, C. T. Management of Hepatocellular Carcinoma: Current Status and Future Directions. Gut Liver 2015, 9, 437–448. (4) Yamauchi, K.; Yang, M.; Hayashi, K.; Jiang, P.; Yamamoto, N.; Tsuchiya, H.; Tomita, K.; Moossa, A. R.; Bouvet, M.; Hoffman, R. M. Induction of Cancer Metastasis by Cyclophosphamide Pretreatment of Host Mice: An Opposite Effect of Chemotherapy. Cancer Res. 2008, 68, 516–520. (5) Wu, J.; Muldoon, L. L.; Dickey, D. T.; Lewin, S.; Varallyay, C. G.; Neuwelt, E. A. Cyclophosphamide Enhances Human Tumor Growth in Nude Rat Xenografted Tumor Models.


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ToC Graphic


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Locally Deployable Nanofiber Patch for Sequential Drug Delivery in

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