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. 2022 May 20;42(5):625–632. doi: 10.12122/j.issn.1673-4254.2022.05.01

An injectable hydrogel/staple fiber composite for sustained release of CA4P and doxorubicin for combined chemotherapy of xenografted breast tumor in mice

持续释放CA4P和阿霉素的可注射水凝胶/短纤维复合物用于小鼠乳腺癌异种移植瘤联合化疗

Ting WANG 1, Ling YANG 1, Yuhan XIE 1, Siyu CHENG 1, Min XIONG 1, Xiaoming LUO 1,*
PMCID: PMC9178649  PMID: 35673904

Abstract

Objective

To prepare an injectable hydrogel/staple fiber composite loaded with combretastain A-4 disodium phosphate (CA4P) and doxorubicin (DOX) and evaluate its antitumor efficacy via intratumoral injection.

Methods

DOX-loaded PELA staple fibers (FDOX) were prepared using electro-spinning and cryo-cutting, and the drug distribution on the surface of the fibers was observed using a fluorescence microscope, and the encapsulation efficiency and loading capacity of FDOX were determined with a fluorospectro photometer. The fibers were then dispersed in CA4P-loaded PLGA-PEG-PLGA tri-block polymer solution at room temperature to obtain the hydrogel/staple fiber composite (GCA4P/FDOX). The thermo-sensitivity of this composite was determined by a test tube inverting method. An ultraviolet spectrophotometer and a fluorospectrophotometer were used to detect the release profile of CA4P and DOX, respectively. We observed in vivo gel formation of the composite after subcutaneous injection in mice. The in vitro cytotoxicity of GCA4P/FDOX composite in MCF-7 and 4T1 cells was assessed using cell Counting Kit-8 (CCK-8) reagent. In a mouse model bearing breast tumor 4T1 cell xenograft, we evaluated the antitumor efficacy of the composite by monitoring tumor growth within 30 days after intratumoral injection of the composite. HE staining, immunohistochemistry for Ki67 and immunofluorescence (TUNEL) assay were used for pathological examination of the tumor tissues 21 days after the treatments.

Results

The average length of FDOX was 4.0±1.3 μm, and its drug loading capacity was (2.69±0.35)% with an encapsulation efficiency of (89.70±0.12)%. DOX was well distributed on the surface of the fibers. When the temperature increased to 37 ℃, the composite rapidly solidified to form a gel in vitro. Drug release behavior test showed that CA4P was completely released from the composite in 5 days and 87% of DOX was released in 30 days. After subcutaneous injection, the composite solidified rapidly without degradation at 24 h after injection. After incubation with GCA4P/FDOX for 72 h, only 30.6% of MCF-7 cells and 28.9% of 4T1 cells were viable. In the tumor-bearing mice, the tumor volume was 771.9±76.9 mm3 in GCA4P/FDOX treatment group at 30 days. Pathological examination revealed obvious necrosis of the tumor tissues and tumor cell apoptosis induced by intratumoral injection of G4A4P/FDOX.

Conclusion

As an efficient dual drug delivery system, this hydrogel/staple fiber composite provides a new strategy for local combined chemotherapy of solid tumors.

Keywords: electro-spinning, staple fibers, doxorubicin, CA4P, drug delivery system

INTRODUCTION

Tumor blood vessels play a key role in tumor progression and metastasis, but they also provide an important therapeutic target for treatment of malignant solid tumors [1]. Currently two types of therapeutic agents targeting tumor vasculature are available, namely anti-angiogenic agents and vascular disrupting agents (VDAs) [2]. Several anti-angiogenic agents such as bevacizumab, sunitinib and sorafenib, which inhibit the formation of new blood vessels, have been used clinically for treating malignant tumors, but their effects are substantially affected by tumor types and tumor heterogeneity[3], and as a common problem in cancer treatment, these agents may also cause tumor resistance to lower their therapeutic benefits[4].

VDAs have been shown to have lower tumor resistance than the anti-angiogenic agents[5]. Among the VDAs, combretastain A-4 disodium phosphate (CA4P) shows a promising anti-vascular effect by specifically destabilizing the microtubule in the endothelial cells of the tumor [6]. Nevertheless, monotherapy with CA4P causes hypoxia and necrosis in center of the tumor but may leave tumor residue at the periphery, thus leading to a potential risk of tumor recurrence[7]. Pre-clinical studies demonstrated that CA4Ps showed an enhanced antitumor effect when combined with other approaches such as antiangiogenic agents, chemotherapy, radiotherapy and radio-immunotherapy [8]. In addition, the conventional systemic administration of CA4Ps almost inevitably causes cardiotoxicity with relatively low drug enrichment at the tumor site.

The emergence of controlled drug delivery systems have offered a new strategy for cancer therapy, which allows sustained maintenance of an local effective therapeutic concentration at the tumor site, thus reducing adverse effects and improving the efficacy of the drug[9] Compared with the controlled drug delivery systems that release a single drug, the dual or multidrug delivery systems with distinct release profiles are capable of both temporally and concentration-dependent release of different molecules to mimic the natural biological events [10]. Inspired by this concept, VDAs and cytotoxic drugs are often entrapped in the same drug vehicles to achieve combined cancer treatment, using the nanocarriers including nanocapsules, polymer nanoparticles and other nanoplatforms[11, 12]. But given the symmetrical geometric structure of the nano-carriers and the diverse physiochemical properties of the drugs, these drug delivery systems can not achieve precise drug loading ratio and release schedule[13]. In addition, as most of therapeutic nanoparticles for solid tumor treatment are administered systemically, their targeting efficacy can be influenced by their interaction with the proteins in blood, the extravasation into and interaction with the perivascular tumor microenvironment (TME) and the properties of the nanoparticles[14].

Local drug delivery systems such as preformed implantable scaffolds and injectable soft materials can achieve higher drug accumulation by delivering the drug directly to the tumor tissues. Up to now, two categories of local drug delivery systems have been used for in situ tumor therapy: one is the well-established drug-loaded implant fabricated by electrospinning, casting or 3D printing, and the other is in situ formed implants such as gels with special responsiveness to either temperature or pH of the tumor microenvironment[15]. In particular, the themo-sensitive hydrogels contain both hydrophobic and hydrophilic components, which can undergo phase transition or swelling/de-swelling as the ambient temperature changes[16]. Various synthetic poly-mers such as poly(N-isopropylacrylamide) (pNIPAAM) [17], poly (ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) and PEG-poly((lactic acid) co-(glycolic acid))-PEG (PEG-PLGA-PEG) ABA or BAB-type triblock copolymers have been widely used to prepare thermo-sensitive gels for injection at the tumor site[18, 19]. A wide range of chemotherapy drugs can be encapsulated in these injectable gels for single or dual drug delivery[20]. Yu et al fabricated a hydrogel based on methoxy poly(ethylene glycol)-poly(r-ethyl-Lglutama-te) (mPEG-b-PELG) polymer co-loaded with CA4P and cisplatin (CDDP) and achieved prolonged release of both CA4P and CDDP[21]. More importantly, the combination of hydrogels with dispersed drug-loaded nanoparticles not only enhances intratumoral drug retention but also achieves moderate drug delivery, thus producing an excellent tumor-suppressing effect[22, 23]. Various nanoparticles can be embedded in hydrogels for local injection.

Compared with spherical nanoparticles, the nanoparticles with non-spherical shapes (nanorods and filamentous or worm-like micelles) can be more easily captured by tumor extracellular matrix (ECM) and the perivascular space close to leaky blood vessels to increase drug accumulation at the intermediate sites[24]. To achieve maximum drug retention at the tumor site, we fabricated a doxorubicin hydrochloride (DOX)-loaded staple electrospun fiber with a length of 4 μm and dispersed the fibers in amphiphilic poly (D, L-lactideco-glycolide)-b-poly(ethylene-glycol)-b-poly (D, L-lactide-co-glycolide) (PLGA-PEG-PLGA) tri-block polymer solution to obtain a CA4P-loaded gel/CA4Ploaded staple fiber composite. This mixture can transform into hydrogel after intratumoral injection and achieve sustained release of CA4P and DOX. CA4P released from the hydrogel first shut down the blood vessels of the tumor, and the subsequently released DOX from the staple fibers can gain access into the interior of the tumor, thus increasing the drug enrichment in the tumor cells and induce cell apoptosis.

METHODS

Materials and reagents

PLGA-PEG-PLGA tri-block copolymer (relative molecular mass of 5250, 1800-1500-1800, LA∶GA=3∶1) was purchased from Jinan Daigang Biomaterial Co. Ltd (Jinan, Shandong). Copolymer poly(ethylene glycol)- polylactide (PELA, Mw=42 300, Mw/Mn=1.23) containing 10% poly(ethylene glycol) (PEG) was synthesized as described previously[25]. Doxorubicin hydrochloride (DOX ∙ HCl) and CA4P were purchased from Dalian Meilun Biotech Co. Ltd (Dalian, China). Cell Counting Kit-8 reagent (CCK-8) was purchased from Dojindo Laboratories (Japan). Ki67 cell proliferation detection kit (KGA321) and one-step TUNEL cell apoptosis detection kit (KGA7062) were purchased from Jiangsu KeyGen BioTECH Co.Ltd (Nanjing, China). All the other chemicals used in this study were of analytical grade and provided by Chengdu Kelong Chemical Reagent Factory (Chengdu, China).

Preparation of DOX-loaded PELA staple fibers

DOX ∙ HCl was dechlorinated with triethylamine before electrospinning. DOX-loaded PELA staple fibers were prepared by electro-spinning combined with cryo-cutting as described previously[26]. Briefly, 30 mg DOX solution in 200 μL DMSO was added into 1g PELA solution in chloroform to obtain a 15% PELA solution by gently stirring the mixture using a magnetic stirrer (FlatSpin, Shanghai Shibin Electronic Technology Co. Ltd). For electrospinning, the polymer solution containing DOX was transferred to a 5 mL syringe fixed on a microinjection pump (Zhejiang University Medical Instrument Co., Hangzhou, China) with the flow rate set at 0.15 mL/h. The fibers were collected on a piece of aluminum foil wrapped on a grounded rotating mandrel. After vacuum drying for 48 h, the fibrous mats were collected and folded perpendicularly to fiber alignment and frozen rapidly in liquid nitrogen. The frozen fibers were sectioned immediately using a cryostat microtome (LEICA1860, Leica Biosystems, Germany) at -20 ℃ at the thickness of 5 μm to obtain DOX-loaded staple fibers (FDOX). The fibers were then washed for 3 times with double distilled water and freeze-dried. The blank fibers (BF) were prepared following the same procedure.

Preparation of drug-loaded hydrogel/staple fiber composite

To fabricate the hydrogel/staple fiber composite, we first prepared CA4P-loaded hydrogel. Briefly, CA4P (15 mg) and PLGA-PEG-PLGA (100 mg) were dispersed together in 5 mL of PBS under continuous stirring at 25 ℃ until a clear solution was obtained (GCA4P). FDOX fibers (20 mg) were then dispersed into 25 mL of GCA4P solution to prepare the drug-loaded hydrogel/staple fiber composite with a theoretical CA4P to DOX ratio of 5∶1 (m/m). To facilitate homogeneous dispersion of the staple fibers in PLGA-PEG-PLGA copolymer solution, 2% sodium alginate was added. After ultra-sonication for 10 min, the hydrogel/staple fiber composite loaded with both CA4P and DOX (GCA4P/FDOX) was obtained and stored at 4 ℃ for later use.

Characterization of the composite

We measured the length of the staple fibers on optical microscopic images (×400 magnification) and calculated the average value using an e-ruler. The length distribution was determined by calculating the ratios of the staple fibers with different lengths in every 100 staple fibers in 5 different areas on inverted phase-contrast microscopic images (Olympus CK40, Japan). The distribution of DOX in FDOX was observed using a fluorescence microscope (Olympus IX71, Japan). The encapsulation efficiency and loading amount of FDOX were determined using a fluorospectrophotometer (Hitachi F-7000, Japan).

In vitro thermo-sensitivity test of the composite

The thermo-sensitivity of this composite was determined using the test tube inverting method[27]. Briefly, 20 mg of FDOX fibers were dispersed into 25 mL of aqueous solution containing both CA4P and PLGA-PEG-PLGA polymer, and the mixture was incubated at 37 ℃ for 15 min. The specimen was considered to form a gel until no visible flow was observed, and photographs before and after solidification were taken with a digital camera.

In vitro drug release from GCA4P/FDOX composite

In vitro release of CA4P and DOX from the composite was characterized by incubating a dialysis bag containing 2 mL of GCA4P/FDOX composite in a 50-mL centrifuge tube in a water bath at 37 ℃ until hydrogel formation. After that, 40 mL of PBS was added into each tube and all the samples were shaken at 100 r/min. At predetermined time points, 1 mL of the release liquid was withdrawn, and 1 mL of fresh PBS was added in the tube for further incubation. The amount of CA4P and DOX release was measured using an ultraviolet spectrophotometer [UV- 5800 (PC), Chengdu Century Ark Technology Co., Ltd] and a fluorospectrophotometer, respectively.

In vitro cytotoxicity test

We tested in vitro cytotoxicity of GCA4P/FDOX composite using Cell Counting Kit-8 (CCK-8) reagent in human breast cancer MCF-7 cells and mouse breast cancer 4T1 cells, provided by the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in our laboratory. Briefly, the cells in logarithmic growth phase were seeded in 48-well plates at a density of 1× 104 cells per well. After incubation for 24 h to allow cell attachment, the sterilized samples of GCA4P/FDOX, FDOX, blank hydrogel (BG) and blank fiber (BF) were added in the wells. The doses of CA4P and DOX in GCA4P/FDOX was equivalent to 5.0 μmol/L and 1.0 μmol/L, respectively. The cells without any treatment were used as the control. After the cells were incubated with the samples for 24, 48 and 72 h, the medium and samples were carefully removed. The cells in each well were washed for 3 times with PBS and then incubated with 300 μL of fresh RPMI 1640 culture medium and 30 μL of CCK-8 reagent, with 300 μL of fresh RPMI 1640 containing 30 μL of CCK-8 reagent but without cells as the blank control. After 4 h of incubation, 100 μL of incubation solution was pipetted from each well and transferred into a new 96-well plate for measurement. The absorbance at 450 nm of each well was determined using an automated microplate spectrophotometer (Multiskan GO1510, Thermo Fisher Scienticic Co., Ltd. USA.) and the relatively cell viability was calculated.

Assessment of in vivo gel formation

All animal experiments were carried out in accordance with the Guide for the Care and the Use of Laboratory Animals as promulgated by the National Institute of Health. Three male Balb/c mice (6-8 weeks old, 18-22 g) were used for in vivo gel formation test of the composite. The hair on the right back of each mouse was removed with 10% Na2S before subcutaneous injection of 100 μL of PBS, FDOX or GCA4P/FDOX suspensions, and at 12 and 24 h after the injection, the volume of the pimple at the injection site was qualitatively monitored and photographed with a digital camera.

Assessment of in vivo antitumor effect of the composite

We used a female Balb/c mouse model bearing subcutaneous mouse breast cancer 4T1 cell xenograft to investigate the antitumor effect of GCA4P/FDOX composite. At 7 days after tumor cell inoculation, the tumors reached a volume of ~50 mm3. The tumor-bearing mice were randomly divided into 3 groups (n=8 per group) for intratumoral injections of 200 μL of normal saline, FDOX, or GCA4P/FDOX. The doses of CA4P and DOX were equivalent to 20.0 and 4.0 mg/kg, respectively. The tumor volume was calculated by measuring the length and width of tumors with a Vernier caliper, and the body weight was also monitored for 30 days. On day 14, the tumors were dissected and fixed in 10% neutral formaldehyde, and then embedded in paraffin blocks and sectioned at a thickness of 4 μm. The obtained tissue sections were stained with hematoxylin and eosin (HE). To investigate the proliferation and apoptosis of the tumor cells, immunohistochemistry (IHC) for Ki-67 and tetramethylrhodamine (TRITC)-labeled terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were performed following the manufacturer's instructions, and the images of HE staining and IHC were obtained with a light microscope (Nikon Eclipse E400, Japan), and TUNEL staining was observed using a fluorescence microscope. The rates of cell proliferation and apoptosis were calculated using Image J software.

Statistics analysis

All the experiments were performed at least in triplicates and the data are presented as Mean±SD. Student's t-test was used for comparisons between two groups, and One-way Analysis of Variance (ANOVA) was performed for comparisons of more than two groups. A probability value (P) of less than 0.05 was considered to indicate a statistically significant difference.

RESULTS AND DISCUSSION

Characterizations of hydrogel/staple fiber composite

As shown in Fig. 1A, the length of FDOX was mainly distributed between 3 μm and 5 μm, and the fibers 4 μm in length accounts for 76%; the average length of FDOX was 4.0±1.3 μm. Due to the spontaneous fluorescence characteristic of DOX, we observed the drug distribution with a fluorescence microscope. Fig. 1B shows no apparent aggregation of the staple fibers, and DOX is homogeneously distributed on the fibers. To ensure the good antitumor efficacy of the fibers, we tested the loading amount and the encapsulation efficiency of DOX. The loading amount of DOX was (2.69±0.35)% and the encapsulation efficiency was (89.70±0.12)% in FDOX. There was about 10% of drug loss, possibly as a result of a small amount of drug release from the fiber surface when soaking the fiber in water during the cryo-cutting process.

1.

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Characterizations of the drug-loaded hydrogel/staple (GCA4P/FDOX) composite. A: Size distribution of staple fibers calculated under light microscopy. B: Fluorescence image of DOX-loaded PELA staple fibers. C, D: In vitro sol-gel state of the composite at 25 ℃ and 37 ℃.

PLGA-PEG-PLGA solution is in a sol state at room temperature and transforms into a water insoluble gel at body temperature due to the hydrophobic interaction between PEG chains at 37 ℃[28, 29]. As shown in Fig. 1C, at room temperature, the staple fibers were well dispersed in PLGA-PEG-PLGA polymer solution containing CA4P, which could flow easily. However, when the temperature increased to 37 ℃, the dispersions became solidified and formed hydrogel, and could not flow. These results indicate that the addition of FDOX fibers had no significant effect on the thermo-sensitivity of GCA4P, and the GCA4P/FDOX composite could be used as a potential injectable formulation for local tumor therapy.

In vitro release of CA4P and DOX

The administration schedule was an important factor in combined therapy with VDAs and other chemotherapeutic drugs. Studies suggested that the administration of VDAs prior to chemotherapeutics caused a delay of the latter to reach the tumor 'core', and the effects of the additional chemotherapeutic could be further potentiated through VDAs-mediated 'trapping' of the drug within the tumor [30, 31]. As an excellent thermo-sensitive material, PLGA-PEG-PLGA tri-block copolymer is often used to carry hydrophilic and hydrophobic chemotherapeutic agent for local cancer therapy[32, 33].

As shown in Fig. 2A, an early burst release followed by sustained release of CA4P occurred when incubating GCA4P/FDOX in PBS at 37 ℃. Approximately 64.3% of CA4P was released from the composite within 4 h, and almost complete CA4P release occurred in 5 days. This can be attributed to the fact that CA4P has a high water solubility and its release from PLGA-PEG-PLGA mainly depends on the diffusion-controlled process[34]. However, the release profile of DOX was different from that of CA4P, and no obvious burst release of DOX was detected within 24 h, approximately 87% of DOX was released from GCA4P/FDOX within 30 days. The release mechanism of DOX not only depends on drug diffusion but also on the degradation of the composite matrix[35], thus resulting in a slower release rate of DOX from the GCA4P/FDOX composite than from FDOX (Fig. 2B).

2.

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Accumulative release of CA4P and DOX from GCA4P/FDOX composite in PBS (pH=7.4) at 37 ℃. A: Accumulative release of CA4P from GCA4P/FDOX. B: Accumulative release of DOX from DOX-loaded fibers (FDOX) and GCA4P/FDOX. Data are presented as Mean±SD (n=3).

In vitro cytotoxicity of GCA4P/FDOX

As a water-soluble prodrug, CA4P can rapidly metabolize into combretastatin A4 (CA4)[36]. We previously found that CA4-loaded fibers could improve the antitumor efficacy of chemotherapeutic drugs by inhibiting tube formation of vascular endothelial cells [37]. Here we examined the cytotoxicity of the GCA4P/FDOX composite using CCK-8 assay. As shown in Fig. 3, both MCF-7 cells (Fig. 3A) and 4T1 cells (Fig. 3B) showed no significant reduction of cell viability in the presence of BG or BF even at 72 h, but the cell viability decreased progressively after incubation with GCA4P/FDOX and FDOX for 24, 48, and 72 h. Due to the sustained release of CA4P and DOX, only 30.6% of MCF-7 cells and 28.9% of 4T1 cells still remained viable after incubation with GCA4P/FDOX for 72 h, and these rates were higher than those of cells incubated with FDOX. These results demonstrate an strong tumor inhibition efficacy of GCA4P/FDOX composite in vitro.

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Efficacy of the hydrogel/staple fiber composite for in vitro tumor cell inhibition against MCF-7 cells (A) and 4T1 cells (B) at 24, 48 and 72 h. Data are presented as Mean±SD (n=4). *P < 0.05, **P < 0.01.

In vivo thermo-sensitivity of GCA4P/FDOX composite

In PLGA-PEG-PLGA hydrogel, a proportion of the hydrophobic and hydrophilic groups interact as the temperature changes [38]. Under the lower critical temperature (LCST), the gel swells in aqueous solution, but once the temperature rises to the LCST, the gel shrinks with volume changes[39]. Fig. 4 shows that after subcutaneous injection 100 μL of GCA4P/FDOX suspension, gel formation in situ occurred due to the body temperature of the mice, and the volume of the formed gel at the subcutaneous layer was smaller than that of saline and FDOX. Even at 24 h, no apparent change of the gel volume was observed due to the slow degradation of PLGA-PEG-PLGA polymer.

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In vivo gel formation of GCA4P/FDOX composite after subcutaneous injection in Balb/c mice. Saline and FDOX were used as the control.

Tumor growth inhibition and body weight change

CA4P is one of the most potent VDAs, which can specifically block the existing blood vessels of the tumor and causes necrosis of the tumor tissue. We assessed the antitumor efficacy of intratumoral injection of GCA4P/FDOX composite in a mouse model bearing subcutaneous 4T1 cell xenograft. Fig. 5 shows the changes in tumor volume and body weight of the mice after intratumoral injection of GCA4P/FDOX and FDOX. Compared with those in saline treatment group, tumors in GCA4P/FDOX and FDOX treatment groups grew more slowly. At 30 days, the tumor volume in GCA4P/FDOX treatment group reached 771.9 ± 76.9 mm3, significantly smaller than that in FDOX and saline treatment groups (1235.7±111.2 mm3 and 1751.0±179.4 mm 3, respectively; Fig. 5A). To investigate the adverse effect of local injection of GCA4P/FDOX and FDOX, we monitored body weight changes of the mice within 30 days after the treatments. As shown in Fig. 5B, all the tumor-bearing mice showed weight loss after modeling, and a roughly 10% weight loss was observed in the treatment groups.

5.

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In vivo antitumor efficacy (A) and body weight change (B) within 30 days after intratumoral injection with saline, FDOX and GCA4P/FDOX composite.

Histological, immunochemical and immunofluorescence evaluation

To further evaluate the antitumor efficacy of GCA4P/FDOX, we examined the tumor tissues using HE staining, IHC staining and immunofluorescence assay on day 21 after the treatments. HE staining (Fig. 6A) showed more obvious necrosis in the tumors after treatments with FDOX and GCA4P/FDOX than after saline treatment. GCA4P/FDOX treatment resulted in the largest necrotic area (pink area) along with vacuolar degeneration of the tumor cells. IHC staining and TUNEL assay showed the lowest positivity for Ki-67 and the most apoptosis cells (red fluorescence) in the tumor in GCA4P/FDOX treatment group (Fig. 6B and 6C). Compared with other treatment, GCA4P/FDOX treatment resulted in a cell proliferation rate of (47.2±3.2)% in the tumor cells, significantly lower than those in saline group [(85.4±6.1)%, P < 0.01] and FDOX treatment group [(68.5± 4.1)%, P < 0.05]. GCA4P/FDOX treatment also caused an apoptosis rate of (54.4±3.9)% in the tumor cells, the highest among the 3 groups. These results demonstrate that GCA4P/ FDOX has the strongest ability to inhibit proliferation and induce apoptosis of the tumor cells after local injection in the tumor-bearing mice.

6.

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Pathological examination of the tumors on day 21 after treatment with saline, FDOX and GCA4P/FDOX composite. A: Typical HE staining, immunohistochemical staining for Ki-67 and immunofluorescence staining (TUNEL) of the tumor tissues. B: Percentage of Ki67-positive cells showing proliferative activity. C: Percentage of apoptotic cells stained with TUNEL. Data are presented as Mean±SD (n=3), *P < 0.05, **P < 0.01.

Conclusion

We prepared an injectable hydrogel/staple fiber composite by physically mixing CA4P-loaded PLGA-PEG-PLGA hydrogel with DOX-loaded staple fibers. This dual drug delivery system is thermo-sensitive and can solidify at 37 ℃ to achieve sustained release of CA4P and DOX and enhanced cytotoxicity. After subcutaneous injection in mice, the composite can quickly forms gels in situ. Compared with FDOX, local injection of GCA4P/FDOX composite results in a stronger antitumor efficacy, suggesting its potential as a dual drug delivery system for local chemotherapy of solid tumors.

Biography

王婷，硕士，E-mail: 1730056370@qq.com

Funding Statement

Supported by National Natural Science Foundation of China (81501581) and the Technology Innovation Team of Chengdu MedicalCollege(CYTD19-02)

国家自然科学基金（81501581）；成都医学院科研创新基金团队项目（CYTD19-02）

Contributor Information

Ting WANG (王 婷), Email: 1730056370@qq.com.

Xiaoming LUO (罗 晓明), Email: lxm@cmc.edu.cn.

References

  • 1.Smolarczyk R, Czapla J, Jarosz-Biej M, et al. Vascular disrupting agents in cancer therapy. E Journal Pharmacol. 2021;891:173692. doi: 10.1016/j.ejphar.2020.173692. [Smolarczyk R, Czapla J, Jarosz-Biej M, et al. Vascular disrupting agents in cancer therapy[J]. E Journal Pharmacol, 2021, 891: 173692.] [DOI] [PubMed] [Google Scholar]
  • 2.Liang WJ, Ni YC, Chen F. Tumor resistance to vascular disrupting agents: mechanisms, imaging, and solutions. Oncotarget. 2016;7(13):15444–59. doi: 10.18632/oncotarget.6999. [Liang WJ, Ni YC, Chen F. Tumor resistance to vascular disrupting agents: mechanisms, imaging, and solutions[J]. Oncotarget, 2016, 7 (13): 15444-59.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Martin JD, Seano G, Jain RK. Normalizing function of tumor vessels: progress, opportunities, and challenges. Annu Rev Physiol. 2019;81:505–34. doi: 10.1146/annurev-physiol-020518-114700. [Martin JD, Seano G, Jain RK. Normalizing function of tumor vessels: progress, opportunities, and challenges[J]. Annu Rev Physiol, 2019, 81: 505-34.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Trenti A, Zulato E, Pasqualini L, et al. Therapeutic concentrations of digitoxin inhibit endothelial focal adhesion kinase and angiogenesis induced by different growth factors. Br J Pharmacol. 2017;174(18):3094–106. doi: 10.1111/bph.13944. [Trenti A, Zulato E, Pasqualini L, et al. Therapeutic concentrations of digitoxin inhibit endothelial focal adhesion kinase and angiogenesis induced by different growth factors[J]. Br J Pharmacol, 2017, 174 (18): 3094-106.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Lei X, Chen M, Li X, et al. A vascular disrupting agent overcomes tumor multidrug resistance by skewing macrophage polarity toward the M1 phenotype. Canc Lett. 2018;418:239–49. doi: 10.1016/j.canlet.2018.01.016. [Lei X, Chen M, Li X, et al. A vascular disrupting agent overcomes tumor multidrug resistance by skewing macrophage polarity toward the M1 phenotype[J]. Canc Lett, 2018, 418: 239-49.] [DOI] [PubMed] [Google Scholar]
  • 6.Dey S, Kumari S, Kalainayakan SP, et al. The vascular disrupting agent combretastatin A-4 phosphate causes prolonged elevation of proteins involved in heme flux and function in resistant tumor cells. Oncotarget. 2017;9(3):4090–101. doi: 10.18632/oncotarget.23734. [Dey S, Kumari S, Kalainayakan SP, et al. The vascular disrupting agent combretastatin A-4 phosphate causes prolonged elevation of proteins involved in heme flux and function in resistant tumor cells [J]. Oncotarget, 2017, 9(3): 4090-101.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Tozer GM, Kanthou C, Baguley BC. Disrupting tumour blood vessels. Nat Rev Cancer. 2005;5(6):423–35. doi: 10.1038/nrc1628. [Tozer GM, Kanthou C, Baguley BC. Disrupting tumour blood vessels [J]. Nat Rev Cancer, 2005, 5(6): 423-35.] [DOI] [PubMed] [Google Scholar]
  • 8.Pedley RB, Hill SA, Boxer GM, et al. Eradication of colorectal xenografts by combined radioimmunotherapy and combretastatin a-4 3-O-phosphate. Cancer Res. 2001;61(12):4716–22. [Pedley RB, Hill SA, Boxer GM, et al. Eradication of colorectal xenografts by combined radioimmunotherapy and combretastatin a-4 3-O-phosphate[J]. Cancer Res, 2001, 61(12): 4716-22.] [PubMed] [Google Scholar]
  • 9.Sohail M, Guo WN, Li ZY, et al. Nanocarrier-based drug delivery system for cancer therapeutics: a review of the last decade. Curr Med Chem. 2021;28(19):3753–72. doi: 10.2174/0929867327666201005111722. [Sohail M, Guo WN, Li ZY, et al. Nanocarrier-based drug delivery system for cancer therapeutics: a review of the last decade[J]. Curr Med Chem, 2021, 28(19): 3753-72.] [DOI] [PubMed] [Google Scholar]
  • 10.Zou Y, Brooks JL, Talwalkar V, et al. Development of an injectable two-phase drug delivery system for sequential release of antiresorptive and osteogenic drugs. J Biomed Mater Res B Appl Biomater. 2012;100(1):155–62. doi: 10.1002/jbm.b.31933. [Zou Y, Brooks JL, Talwalkar V, et al. Development of an injectable two-phase drug delivery system for sequential release of antiresorptive and osteogenic drugs[J]. J Biomed Mater Res B Appl Biomater, 2012, 100(1): 155-62.] [DOI] [PubMed] [Google Scholar]
  • 11.Wang Z, Ho PC. A nanocapsular combinatorial sequential drug delivery system for antiangiogenesis and anticancer activities. Biomaterials. 2010;31(27):7115–23. doi: 10.1016/j.biomaterials.2010.05.075. [Wang Z, Ho PC. A nanocapsular combinatorial sequential drug delivery system for antiangiogenesis and anticancer activities[J]. Biomaterials, 2010, 31(27): 7115-23.] [DOI] [PubMed] [Google Scholar]
  • 12.Wang N, Liu CY, Yao WH, et al. A traceable, GSH/pH dual-responsive nanoparticles with spatiotemporally controlled multiple drugs release ability to enhance antitumor efficacy. Colloids Surf B Biointerfaces. 2021;205:111866. doi: 10.1016/j.colsurfb.2021.111866. [Wang N, Liu CY, Yao WH, et al. A traceable, GSH/pH dual-responsive nanoparticles with spatiotemporally controlled multiple drugs release ability to enhance antitumor efficacy[J]. Colloids Surf B Biointerfaces, 2021, 205: 111866.] [DOI] [PubMed] [Google Scholar]
  • 13.Zhang LY, Zhang MJ, Zhou L, et al. Dual drug delivery and sequential release by amphiphilic Janus nanoparticles for liver cancer theranostics. Biomaterials. 2018;18:113–25. doi: 10.1016/j.biomaterials.2018.07.060. [Zhang LY, Zhang MJ, Zhou L, et al. Dual drug delivery and sequential release by amphiphilic Janus nanoparticles for liver cancer theranostics[J]. Biomaterials, 2018, 18: 113-25.] [DOI] [PubMed] [Google Scholar]
  • 14.Shi J, Kantoff PW, Wooster R, et al. Cancer nanomedicine: progress, challenges and opportunities. Nat Rev Cancer. 2017;17(1):20–37. doi: 10.1038/nrc.2016.108. [Shi J, Kantoff PW, Wooster R, et al. Cancer nanomedicine: progress, challenges and opportunities[J]. Nat Rev Cancer, 2017, 17(1): 20-37.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Talebian S, Foroughi J, Wade SJ, et al. Biopolymers for antitumor implantable drug delivery systems: recent advances and future outlook. Adv Mater. 2018;30(31):e1706665. doi: 10.1002/adma.201706665. [Talebian S, Foroughi J, Wade SJ et al. Biopolymers for antitumor implantable drug delivery systems: recent advances and future outlook[J]. Adv Mater, 2018, 30(31): e1706665.] [DOI] [PubMed] [Google Scholar]
  • 16.Yu YB, Cheng Y, Tong JY, et al. Recent advances in thermo-sensitive hydrogels for drug delivery. J Mater Chem B. 2021;9(13):2979–92. doi: 10.1039/D0TB02877K. [Yu YB, Cheng Y, Tong JY, et al. Recent advances in thermo-sensitive hydrogels for drug delivery[J]. J Mater Chem B, 2021, 9(13) 2979-92.] [DOI] [PubMed] [Google Scholar]
  • 17.Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Adv Drug Delivery Rev. 2001;53(3):321–39. doi: 10.1016/S0169-409X(01)00203-4. [Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery[J]. Adv Drug Delivery Rev, 2001, 53(3) 321-39.] [DOI] [PubMed] [Google Scholar]
  • 18.Qin L, Ling GX, Peng FF, et al. Black phosphorus nanosheets and gemcitabine encapsulated thermo-sensitive hydrogel for synergistic photothermal-chemotherapy. J Colloid Interface Sci. 2019;556:232–8. doi: 10.1016/j.jcis.2019.08.058. [Qin L, Ling GX, Peng FF, et al. Black phosphorus nanosheets and gemcitabine encapsulated thermo-sensitive hydrogel for synergistic photothermal-chemotherapy[J]. J Colloid Interface Sci, 2019, 556: 232-8.] [DOI] [PubMed] [Google Scholar]
  • 19.Chan PS, Xian JW, Li QQ, et al. Biodegradable Thermosensitive PLGAPEG-PLGA Polymer for Non-irritating and Sustained Ophthalmic Drug Delivery. AAPS J. 2019;21(4):59. doi: 10.1208/s12248-019-0326-x. [Chan PS, Xian JW, Li QQ, et al. Biodegradable Thermosensitive PLGAPEG-PLGA Polymer for Non-irritating and Sustained Ophthalmic Drug Delivery[J]. AAPS J, 2019, 21(4): 59.] [DOI] [PubMed] [Google Scholar]
  • 20.Wei LY, Chen JJ, Zhao SH, et al. Thermo-sensitive polypeptide hydrogel for locally sequential delivery of two-pronged antitumor drugs. Acta Biomater. 2017;58:44–53. doi: 10.1016/j.actbio.2017.05.053. [Wei LY, Chen JJ, Zhao SH, et al. Thermo-sensitive polypeptide hydrogel for locally sequential delivery of two-pronged antitumor drugs[J]. Acta Biomater, 2017, 58: 44-53.] [DOI] [PubMed] [Google Scholar]
  • 21.Yu SJ, Wei S, Liu L, et al. Enhanced local cancer therapy using a CA4P and CDDP co-loaded polypeptide gel depot. Biomater Sci. 2019;7(3):860–6. doi: 10.1039/C8BM01442F. [Yu SJ, Wei S, Liu L, et al. Enhanced local cancer therapy using a CA4P and CDDP co-loaded polypeptide gel depot[J]. Biomater Sci, 2019, 7(3): 860-6.] [DOI] [PubMed] [Google Scholar]
  • 22.Hou MM, Liu WW, Zhang L, et al. Responsive agarose hydrogel incorporated with natural humic acid and MnO2 nanoparticles for effective relief of tumor hypoxia and enhanced photo-induced tumor therapy. Biomater Sci. 2020;8(1):353–69. doi: 10.1039/C9BM01472A. [Hou MM, Liu WW, Zhang L, et al. Responsive agarose hydrogel incorporated with natural humic acid and MnO2 nanoparticles for effective relief of tumor hypoxia and enhanced photo-induced tumor therapy[J]. Biomater Sci, 2020, 8(1): 353-69.] [DOI] [PubMed] [Google Scholar]
  • 23.Maiti D, Chao Y, Dong ZL, et al. Development of a thermosensitive protein conjugated nanogel for enhanced radio-chemotherapy of cancer. Nanoscale. 2018;10(29):13976–85. doi: 10.1039/C8NR03986K. [Maiti D, Chao Y, Dong ZL, et al. Development of a thermosensitive protein conjugated nanogel for enhanced radio-chemotherapy of cancer[J]. Nanoscale, 2018, 10(29): 13976-85.] [DOI] [PubMed] [Google Scholar]
  • 24.Mao W, Son YJ, Yoo HS. Gold nanospheres and nanorods for anticancer therapy: comparative studies of fabrication, surfacedecoration, and anti-cancer treatments. Nanoscale. 2020;12(28):14996–5020. doi: 10.1039/D0NR01690J. [Mao W, Son YJ, Yoo HS. Gold nanospheres and nanorods for anticancer therapy: comparative studies of fabrication, surfacedecoration, and anti-cancer treatments[J]. Nanoscale, 2020, 12(28): 14996-5020.] [DOI] [PubMed] [Google Scholar]
  • 25.Li XH, Zhang YH, Yan RH, et al. Influence of process parameters on the protein stability encapsulated in poly-dl-lactide-poly (ethylene glycol) microspheres. J Control Release. 2000;68(1):41–52. doi: 10.1016/S0168-3659(00)00235-2. [Li XH, Zhang YH, Yan RH, et al. Influence of process parameters on the protein stability encapsulated in poly-dl-lactide-poly (ethylene glycol) microspheres[J]. J Control Release, 2000, 68(1): 41-52.] [DOI] [PubMed] [Google Scholar]
  • 26.Wei JJ, Luo XM, Chen MH, et al. Spatial distribution and antitumor activities after intratumoral injection of fragmented fibers with loaded hydroxycamptothecin. Acta Biomater. 2015;23:189–200. doi: 10.1016/j.actbio.2015.05.020. [Wei JJ, Luo XM, Chen MH et al. Spatial distribution and antitumor activities after intratumoral injection of fragmented fibers with loaded hydroxycamptothecin[J]. Acta Biomater, 2015, 23: 189-200.] [DOI] [PubMed] [Google Scholar]
  • 27.Liu M, Song X, Wen YT, et al. Injectable thermoresponsive hydrogel formed by alginate-g-poly(N-isopropylacrylamide) releasing doxorubicin-encapsulated micelles as smart drug delivery system. ACS Appl Mater Interfaces. 2017;9(41):35673–82. doi: 10.1021/acsami.7b12849. [Liu M, Song X, Wen YT, et al. Injectable thermoresponsive hydrogel formed by alginate-g-poly(N-isopropylacrylamide) releasing doxorubicin-encapsulated micelles as smart drug delivery system[J]. ACS Appl Mater Interfaces, 2017, 9(41): 35673-82.] [DOI] [PubMed] [Google Scholar]
  • 28.Wei PS, Chen YJ, Lin SY, et al. In situ subcutaneously injectable thermosensitive PEG-PLGA diblock and PLGA-PEG-PLGA triblock copolymer composite as sustained delivery of bispecific anti-CD3 scFv T-cell/anti-EGFR Fab Engager (BiTEE) Biomaterials. 2021;278:121166. doi: 10.1016/j.biomaterials.2021.121166. [Wei PS, Chen YJ, Lin SY, et al. In situ subcutaneously injectable thermosensitive PEG-PLGA diblock and PLGA-PEG-PLGA triblock copolymer composite as sustained delivery of bispecific anti-CD3 scFv T-cell/anti-EGFR Fab Engager (BiTEE)[J]. Biomaterials, 2021, 278: 121166.] [DOI] [PubMed] [Google Scholar]
  • 29.Pelton R. Temperature-sensitive aqueous microgels. Adv Colloid Interface Sci. 2000;85:1–33. doi: 10.1016/S0001-8686(99)00023-8. [Pelton R. Temperature-sensitive aqueous microgels[J]. Adv Colloid Interface Sci, 2000, 85: 1-33.] [DOI] [PubMed] [Google Scholar]
  • 30.Fruytier AC, Le Duff CS, Po C, et al. The blood flow shutdown induced by combretastatin A4 impairs gemcitabine delivery in a mouse hepatocarcinoma. Front Pharmacol. 2016;7:506. doi: 10.3389/fphar.2016.00506. [Fruytier AC, Le Duff CS, Po C, et al. The blood flow shutdown induced by combretastatin A4 impairs gemcitabine delivery in a mouse hepatocarcinoma[J]. Front Pharmacol, 2016, 7: 506.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Gill JH, Rockley KL, Santis CD, et al. Vascular Disrupting Agents in cancer treatment: Cardiovascular toxicity and implications for coadministration with other cancer chemotherapeutics. Pharmacol Ther. 2019;202:18–31. doi: 10.1016/j.pharmthera.2019.06.001. [Gill JH, Rockley KL, Santis CD, et al. Vascular Disrupting Agents in cancer treatment: Cardiovascular toxicity and implications for coadministration with other cancer chemotherapeutics[J]. Pharmacol Ther, 2019, 202: 18-31.] [DOI] [PubMed] [Google Scholar]
  • 32.Wang PX, Chu W, Zhuo XZ, et al. Modified PLGA-PEG-PLGA thermosensitive hydrogels with suitable thermosensitivity and properties for use in a drug delivery system. J Mater Chem B. 2017;5(8):1551–65. doi: 10.1039/C6TB02158A. [Wang PX, Chu W, Zhuo XZ, et al. Modified PLGA-PEG-PLGA thermosensitive hydrogels with suitable thermosensitivity and properties for use in a drug delivery system[J]. J Mater Chem B, 2017, 5(8): 1551-65.] [DOI] [PubMed] [Google Scholar]
  • 33.Yang ZM, Yu SJ, Li DS, et al. The effect of PLGA-based hydrogel scaffold for improving the drug maximum-tolerated dose for in situ osteosarcoma treatment. Colloids Surf B Biointerfaces. 2018;172:387–94. doi: 10.1016/j.colsurfb.2018.08.048. [Yang ZM, Yu SJ, Li DS, et al. The effect of PLGA-based hydrogel scaffold for improving the drug maximum-tolerated dose for in situ osteosarcoma treatment[J]. Colloids Surf B Biointerfaces, 2018, 172: 387-94.] [DOI] [PubMed] [Google Scholar]
  • 34.Cao DLG, Zhang XX, Akabar MD, et al. Liposomal doxorubicin loaded PLGA-PEG-PLGA based thermogel for sustained local drug delivery for the treatment of breast cancer. Artif Cells Nanomed Biotechnol. 2019;47(1):181–91. doi: 10.1080/21691401.2018.1548470. [Cao DLG, Zhang XX, Akabar MD, et al. Liposomal doxorubicin loaded PLGA-PEG-PLGA based thermogel for sustained local drug delivery for the treatment of breast cancer[J]. Artif Cells Nanomed Biotechnol, 2019, 47(1): 181-91.] [DOI] [PubMed] [Google Scholar]
  • 35.Zhang WJ, Xu WG, Ning C, et al. Long-acting hydrogel/microsphere composite sequentially releases dexmedetomidine and bupiva-caine for prolonged synergistic analgesia. Biomaterials. 2018;181:378–91. doi: 10.1016/j.biomaterials.2018.07.051. [Zhang WJ, Xu WG, Ning C, et al. Long-acting hydrogel/microsphere composite sequentially releases dexmedetomidine and bupiva-caine for prolonged synergistic analgesia[J]. Biomaterials, 2018, 181: 378-91.] [DOI] [PubMed] [Google Scholar]
  • 36.Zhou PF, Liang YR, Zhang H, et al. Design, synthesis, biological evaluation and cocrystal structures with tubulin of chiral β-lactam bridged combretastatin A-4 analogues as potent antitumor agents. Eur J Med Chem. 2018;144:817–2. doi: 10.1016/j.ejmech.2017.12.004. [Zhou PF, Liang YR, Zhang H, et al. Design, synthesis, biological evaluation and cocrystal structures with tubulin of chiral β-lactam bridged combretastatin A-4 analogues as potent antitumor agents[J]. Eur J Med Chem, 2018, 144: 817-2.] [DOI] [PubMed] [Google Scholar]
  • 37.Luo XM, Xu GS, Wei JJ, et al. Tunable release of chemotherapeutic and vascular disrupting agents from injectable fiber fragments potentiates combination chemotherpy. Int J Pharm. 2016;506(1- 2):1–12. doi: 10.1016/j.ijpharm.2016.04.037. [Luo XM, Xu GS, Wei JJ, et al. Tunable release of chemotherapeutic and vascular disrupting agents from injectable fiber fragments potentiates combination chemotherpy[J]. Int J Pharm, 2016, 506(1- 2): 1-12.] [DOI] [PubMed] [Google Scholar]
  • 38.Chen CH, Kuo CY, Chen SH, et al. Thermosensitive injectable hydrogel for simultaneous intraperitoneal delivery of doxorubicin and prevention of peritoneal adhesion. Int J Mol Sci. 2018;19(5):1373. doi: 10.3390/ijms19051373. [Chen CH, Kuo CY, Chen SH, et al. Thermosensitive injectable hydrogel for simultaneous intraperitoneal delivery of doxorubicin and prevention of peritoneal adhesion[J]. Int J Mol Sci, 2018, 19(5): 1373.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Sapino S, Chirio D, Peira E, et al. Ocular drug delivery: a special focus on the thermosensitive approach. Nanomaterials (Basel) 2019;9(6):884. doi: 10.3390/nano9060884. [Sapino S, Chirio D, Peira E, et al. Ocular drug delivery: a special focus on the thermosensitive approach[J]. Nanomaterials (Basel), 2019, 9 (6): 884.] [DOI] [PMC free article] [PubMed] [Google Scholar]

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