Enhanced local cancer therapy using a CA4P and CDDP co-loaded polypeptide gel depot

Shuangjiang Yu ad, Shu Wei b, Liang Liu a, Desheng Qi ab, Jiayu Wang ab, Guojun Chen c, Wanying He a, Chaoliang He *a, Xuesi Chen a and Zhen Gu *cd
aKey Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China. E-mail: clhe@ciac.ac.cn
bSchool of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022, P. R. China
cDepartment of Bioengineering, California Nanosystems Institute (CNSI), Jonsson Comprehensive Cancer Center, and Center for Minimally Invasive Therapeutics, University of California, Los Angeles (UCLA), 90095, USA. E-mail: guzhen@ucla.edu
dJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, USA

Received 9th November 2018 , Accepted 19th January 2019

First published on 23rd January 2019

Cancer combination therapy based on drug co-delivery systems provides an effective strategy for enhancing treatment efficacy and reducing side effects. In this work, a new strategy through co-delivery of combretastatin A4 disodium phosphate (CA4P) and cisplatin (CDDP) was developed for the local treatment of colon cancer, through an in situ thermo-gelling hydrogel (mPEG-b-PELG). The results indicated that this material possessed concentration-dependent thermogelling properties and tunable in vivo biodegradability. Also, the drug loaded gel could regulate the in vitro drug release behaviors of both CDDP and CA4P, which promoted the in vivo vessel disrupting effects of CA4P compared with a free drug after local treatment for 48 h. Although the drug co-loaded gel induced less in vitro cell death compared with the free drug co-treated group, this drug co-loaded gel depot showed the highest antitumor efficacy compared with the other experimental groups after peritumoral injection toward C26 tumor bearing mice.

1. Introduction

Although chemotherapeutic agents have been widely utilized for cancer treatment, several major drawbacks still limit their clinical outcomes, such as non-specific distribution in vivo, drug resistance and undesirable side effects.1–6 Using drug delivery systems based on both macroscale and particulate formulations has been demonstrated to be an efficient approach to enhance antitumor efficacy with the capabilities of regulating drug release behavior and drug biodistribution.7–12 Among them, injectable hydrogels have been widely engineered for controlled drug release toward localized cancer treatments.13–17 In particular, thermo-gelling hydrogels, which can undergo a sol-to-gel transition under the stimulus of body temperature after local administration, show practical advantages for both scientific research and clinical applications.18–20 Nowadays, ample therapeutics have been delivered using thermo-responsive hydrogels for improving antitumor efficacy, including small chemotherapeutic drugs,21–23 bioactive macromolecules,8,18,24 and radioisotopes.25,26

Besides the improvements of the administration route, combination therapy based on different types of anticancer therapeutics has been proven to be an effective method for decreasing the clinical drug dosage, reducing the untoward side effects, and maximizing the drug efficacy.27–30 For example, combretastatin A4 disodium phosphate (CA4P) is a tubulin-binding tumor vascular disrupting agent (VDA) with admirable aqueous solubility that exhibited selective toxicity towards the vasculature of solid tumors.31–33 However, similar to other small-molecule VDAs, a key challenge is that CA4P can typically cause central tumor hemorrhagic necrosis, but living tumor cells can still exist in the tumor periphery because of the continuous supply of oxygen and nutrients from blood vessels in normal tissues.33,34 Therefore, the combination of CA4P and chemotherapeutic agents may provide an effective approach for enhanced tumor inhibition.33,35,36 Another potential limitation of CA4P is the rapid dissociation behavior with tubulin which could lead to the restitution of blood flow within 24 h following the treatments in some tumor models.32 Furthermore, dose-limiting toxicities (DLTs) of CA4P were also observed in clinical trials, such as tumor-induced pain, syncope, dyspnea, and coronary vasospasm, which largely limit its further applications.37,38 Exploring the CA4P containing local drug co-delivery system might therefore have potential to overcome the above challenges.

In our previous work, a series of poly(L-glutamate)-based thermo-gelling hydrogels were exploited for cell culture and localized drug delivery.21,22,24,39 Herein, a thermo-gelling hydrogel based on methoxy poly(ethylene glycol)-poly(γ-ethyl-L-glutamate) diblock copolymers (mPEG-b-PELG) was prepared and employed to engineer the local CA4P and CDDP co-delivery system for treatment of mouse colon cancer (C26) (Fig. 1). The in vitro drug release behavior and in vivo time-dependent tumor vessel disrupting behaviors regulated by the drug-loaded hydrogel were evaluated in detail. Also, the antitumor efficacy and safety of this drug co-loaded gel depot were investigated in the BALB/c mice bearing C26 xenograft models.

image file: c8bm01442f-f1.tif
Fig. 1 Illustration of the local co-delivery of CA4P and CDDP based on a thermo-sensitive polypeptide hydrogel. (A) Structures of the mPEG-b-PELG polymer and drugs. (B) The schematic of the mechanism of in situ co-treatment of CDDP and CA4P through a gel scaffold.

2. Experimental methods

2.1 Preparation and characterization of mPEG-b-PELG

The described polypeptide was prepared according to our previous work with modification.40 Briefly, after eliminating water by a co-boiling method, mPEG-NH2 was re-dissolved in anhydrous DMF. Then, γ-ethyl-L-glutamate N-carboxyanhydride (ELG-NCA) was added into the above flask with N2 protection. The reaction mixture was stirred under the N2 atmosphere for 72 h at 28 °C. The mixture solution was then transferred into a dialysis bag (MWCO: 3.5 kDa) against deionized water for 72 h at room temperature to remove the organic solvent and unreacted monomers. mPEG-b-PELG was finally obtained through lyophilization. The structure and molecular weight were confirmed using 1H NMR spectra recorded on a Bruker AV 400 NMR spectrometer using trifluoroacetic acid-D as a solvent. The mouse colon cancer cell line (C26), human breast cancer cell line (MCF-7), and human umbilical vein endothelial cell line (HUVEC) were obtained from Shanghai Bogoo Biotechnology Co. Ltd, China.

2.2 Gel formation, micro-morphology and rheological property characterization

Different weights of mPEG-b-PELG were dissolved in PBS (pH = 7.4) and stirred for 48 h in an ice-water bath. Then, these copolymer solutions (300 μL) were moved into several small vials (inner diameter: 8 mm). A test tube inverting method was employed to investigate the phase diagram of mPEG-b-PELG with an increase in temperature of 2 °C per step every 10 min. When no fluidity was observed in 0.5 min, the temperature was recorded as the sol-to-gel transition point. In this section, triplicate tests were used to confirm each temperature point. The microstructure of the blank gel was investigated by scanning electron microscopy (SEM, Philips XL 30, 10 kV), for which the sample (8.0 wt%) was dried using a freeze dryer after freezing in liquid nitrogen. The rheological properties of the hydrogel with or without drugs were investigated on a rheometer (Anton Paar, MCR 301) during the sol-to-gel transitions. Briefly, 300 μL of the mPEG-b-PELG solution (8.0 wt%) was pipetted onto a parallel plate (diameter, 25 mm) with a gap of 0.5 mm. The data were collected at a frequency of 1 rad s−1 and a controlled strain γ of 1% with a heating rate of 0.5 °C min−1.

2.3 Study of hydrogel degradation both in vitro and in vivo

500 μL of mPEG-b-PELG solution (8.0 wt%) was added into a vial (inner diameter: 16 mm), which was kept stationary at 37 °C for 10 min. Thereafter, the vial was moved into a shaker at 37 °C. The medium in the vial was replaced with fresh PBS at preset interval points and the remaining mass of the hydrogel was recorded each time. Female Sprague-Dawley (SD) rats (∼200 g) was obtained from the Laboratory Animal Center of Jilin University. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Jilin University and approved by the Animal Ethics Committee of Jilin University. 400 μL of mPEG-b-PELG solution (8.0 wt%) was injected into the right abdomen of rats for evaluating the in vivo hydrogel degradation. The status of residual gel was recorded after sacrificing the rats at predesigned time intervals.

2.4 Drug loading and in vitro drug release

Both CDDP and CA4P were dissolved in the mPEG-b-PELG solution at 4 °C. The final concentrations of the CDDP, CA4P, and copolymer were fixed at 0.6 mg mL−1 (CDDP), 6.0 mg mL−1 (CA4P), and 8.0 wt%, respectively. Then 300 μL of the drug containing solution was transferred into each vial (inner diameter: 8 mm). After the sol-to-gel transition at 37 °C, 2.0 mL of PBS (pH = 7.4) containing 0.1 wt% sodium azide (NaN3) as the drug release medium was added into the above vials. The parallel samples (n = 3) were incubated in an orbital shaker at 37 °C. The drug release medium (1.0 mL) was collected and a fresh one was then added into the vials at the preset time intervals. Each collected drug-containing sample was divided into two parts and kept in 2.0 mL EP-tubes in the dark at room temperature. The CDDP amounts of all the collected samples were determined by ICP-MS (X series II, Thermo Scientific, USA). The released CA4P amount was confirmed using a UV-vis spectrophotometer (PerkinElmer Lambda 365).

2.5 Cytotoxicity evaluation in vitro

C26, MCF-7, and HUVEC cell lines were used to evaluate the in vitro cytotoxicity of the blank mPEG-b-PELG gel, free drug and drug-loaded gel samples by MTT assay. Briefly, the cells (20 × 103 cells per well) were seeded in 24-well plates with 1.0 mL of culture medium. The solutions (50 μL) of the blank gel, CDDP-loaded gel, CA4P-loaded gel, and CDDP and CA4P co-loaded gel were added to a Transwell® insert chamber and changed into hydrogels after incubating for 15 min at 37 °C. Then the chamber was inserted into the cell-containing 24-well plate with fresh culture medium. The cytotoxicity of free CDDP and CA4P was also evaluated using the same method. The MTT assay was performed after 72 h.

2.6 Tumor models

BALB/c mice (female, 5–6 weeks old) were purchased from Beijing Huafukang Biological Technology Co. Ltd. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Jilin University and approved by the Animal Ethics Committee of Jilin University. 2.0 × 106 C26 cells in 100 μL of PBS were inoculated into the backs of BALB/c mice for preparing the xenograft tumor models. The following experiments were performed in detail when the average tumor volume exceeded ∼110 mm3 or ∼370 mm3, respectively.

2.7 Study of the CA4P effect on the microenvironment after local treatments

100 μL of the CA4P containing PBS solution (CA4P dosage: 30 mg kg−1) with or without a copolymer was implanted into the tumor site through a single peritumoral injection when the average tumor volume reached 370 mm3. The external photographs of the tumors were collected at preset intervals. After 48 h, the mice were sacrificed and the tumors were collected and stored in 4.0% paraformaldehyde (PFA) at 4 °C. Then, the paraffin sections of the tumor tissues were stained by the haematoxylin and eosin stain (H&E stain) method and images were observed via a fluorescence microscope (Nikon TE2000-U).

2.8 In vivo antitumor assay

The tumor-bearing mice were randomly divided into 6 groups at the aimed tumor volumes. Different drug formulations with a final volume of 100 μL were peritumorally injected into the tumor sites. Then, the tumor volume and body weight of the treated mice were monitored every two days. Formula (1) was employed to calculate the tumor volume.
image file: c8bm01442f-t1.tif(1)
where a and b represent the length and width of the tumor.

2.9 Histology and TUNEL assay

The treated mice were sacrificed at the 14th day (∼110 mm3) and 12th day (∼370 mm3) after localized treatments with different agents, respectively. Then, the tumor tissues (∼370 mm3) were collected for further studies. The tumor sections were cut into 5 μm thick slices and stained with hematoxylin–eosin (H&E) for histological analysis. Futhermore, the cryogenic slices of the tumor tissues were also made and stained by the terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assay according to our previous work.22 The images of the stained samples were observed on a fluorescence microscope (Nikon TE2000-U).

3. Results and discussion

3.1 mPEG-b-PELG synthesis and characterization

mPEG-b-PELG was prepared through ring-opening polymerization as we previously reported (Fig. S1).40 The degree of polymerization (DP) of poly(L-glutamate) was calculated to be 13 according to 1H NMR spectra (Fig. S2).

3.2 Gelation behavior and properties of mPEG-b-PELG

The sol-to-gel phase transition behavior of mPEG-b-PELG was investigated by the tube inverting method.22 As shown in Fig. 2A, the gelation temperature showed an obvious decrease with the increase of the polymer concentration at pH 7.4. The polymer solution with 8.0 wt% turned into a stable gel phase at around 22 °C, which was considered to be an ideal polymer concentration for further study in this work. The SEM image indicates that the gel (8.0 wt%) had an open porous network, and the mesh size was beyond several tens of micrometers which benefited the drug diffusion and release (Fig. 2B).13 Furthermore, the rheological properties of mPEG-b-PELG (8.0 wt%, pH = 7.4) with or without drugs indicated that the addition of drugs showed no obvious influence on the mechanical properties of the hydrogel (Fig. 2C). Our previous work has demonstrated that the mPEG-b-PELG-based hydrogel exhibited good biocompatibility and biodegradability.24 In this work, the degradation properties of mPEG-b-PELG were further confirmed in detail. As shown in Fig. 2D, only about 35% of the hydrogel (8.0 wt%, 300 μL) disappeared at day 28 in PBS (pH = 7.4). However, the degradation rate was obviously enhanced in vivo, and the gel (8.0 wt%, 400 μL) was completely degraded in 4 weeks under the rat skin (Fig. 2E). Additionally, compared to our previous work,24 the degradation time of this hydrogel in vivo was prolonged for about one week when the polymer concentration increased from 6.0 wt% to 8.0 wt% (Fig. 2E). This tunable in vivo degradation property might be beneficial for its further biomedical applications.
image file: c8bm01442f-f2.tif
Fig. 2 Functional characterization of the mPEG-b-PELG hydrogel. (A) The sol-to-gel phase transition of the mPEG-b-PELG solution with different concentrations. (B) The SEM image of the mPEG-b-PELG formed hydrogel at a concentration of 8.0 wt% (scale bar: 200 μm). (C) The rheology test of the mPEG-b-PELG formed hydrogel (8.0 wt%) with or without drugs. (D) The degradation behavior of the gel (8.0 wt%) in PBS (pH = 7.4) (n = 3). (E) In situ formation and degradation behavior of the hydrogel (8.0 wt%) in vivo.

3.3 Drug release behavior

The release behaviors of CA4P and CDDP from the hydrogel were investigated at 37 °C in PBS (pH = 7.4) in vitro. As shown in Fig. 3A, about 82% of CA4P was released from the gel depot in the first two days, and the final release amount was 90% at day 7. However, the loaded CDDP showed a slower drug release behavior compared with CA4P during the monitoring period, and the release amount was 66% at day 7. These results might be due to the different solubilities of the two drugs. It is worth noting that certain degrees of burst drug release of both CA4P and CDDP were observed in the first several hours, likely due to the fast diffusion of these two water soluble drugs from the gel depot which had a micron-sized porous structure.13
image file: c8bm01442f-f3.tif
Fig. 3 (A) The drug release behavior of CA4P and CDDP from the hydrogel in vitro (pH = 7.4, 37 °C) (n = 3). The viabilities of (B) C26, (C) MCF-7, and (D) HUVEC cells incubated with (+) or without (−) CDDP (3.0 μg mL−1), CA4P (30.0 μg mL−1), and gel (50 μL) for 72 h (n = 3).

3.4 Evaluation of cytotoxicity in vitro

The cytotoxicities of polypeptide hydrogels with or without drugs were investigated against C26, MCF-7, and HUVEC cells. As shown in Fig. 3B–D, the results suggested that the blank gel exhibited good in vitro cytocompatibility. The CDDP-loaded hydrogel exhibited a lower cytotoxicity against HUVEC cells compared with both C26 and MCF-7 cells which indicated that C26 and MCF-7 cell lines were more sensitive to cisplatin compared with the HUVEC cell line. On the other hand, the CA4P-loaded hydrogel showed a higher cytotoxicity against HUVEC cells compared with both C26 and MCF-7 cell lines which demonstrated the selective cytotoxicity of this microtubule depolymerizing agent to endothelial cells.41 It is worth noting that the drug co-loaded gel induced less cell death compared with the free CDDP and CA4P co-treated group, probably due to the prolonged drug release from the gel depot. However, contributing to the drug combination therapy, enhanced tumor cell inhibition effects were observed from the drug co-loaded gel compared with the single drug-loaded gel treated groups.

3.5 CA4P effect on the tumor microenvironment

The primary action of CA4P is selectively targeting endothelial cells, inducing regression of unstable nascent tumor neovessels and hemorrhagic necrosis within the tumor.33,41,42 Herein, the time-dependent intratumoral hemorrhaging behaviors of CA4P alone or CA4P-loaded hydrogel were investigated through peritumoral injection in situ. According to the tumor photographs, slightly enhanced subcutaneous hemorrhage was observed in the free CA4P treated tumor bearing mice compared with the CA4P-loaded gel treated ones after 6 h post-injection. However, enhanced subcutaneous hemorrhage emerged in the tumor treated with the CA4P-loaded gel after 48 h, while no obvious progress of the free CA4P treated one was detected (Fig. 4A). Furthermore, after treating with saline, free CA4P, and CA4P-loaded gel, respectively, for 48 h, the H&E staining images of the tumors indicated that the CA4P-loaded gel led to relatively persistent intratumoral hemorrhage compared with the other two groups (Fig. 4B). These results indicated that the gel depot could enhance the tumor vascular disrupting effects via the sustained release of CA4P.
image file: c8bm01442f-f4.tif
Fig. 4 Characterization of CA4P induced tumor microenvironment changes after local treatments. (A) The photographs of tumors in the red circle after free CA4P and CA4P@Gel injection at 0 h, 6 h, and 48 h (CA4P dosage: 30.0 mg kg−1). (B) H&E staining of the harvested tumors after treating with saline (a), free CA4P (b), and CA4P@Gel (c) at 48 h. The yellow arrows indicate the hemorrhage sites (scale bar: 100 μm).

3.6 Combination antitumor efficacy in vivo

Previous studies have demonstrated that one of the major challenges for the conventional anticancer therapies is controlling the bulky tumor growth.43–46 It has also been verified that CA4P was a desirable candidate for locally combined cancer treatments.36 Thus, in this work, C26 bearing mice with two different sets of initial tumor sizes (around 110 mm3 and 370 mm3, respectively) were employed to evaluate the combination antitumor efficacy. Mice were treated with saline (G1), blank gel (G2), CA4P@Gel (G3), CDDP@Gel (G4), free CA4P and CDDP (G5), or CA4P&CDDP@Gel (G6) through in situ injection at the first treatment day. The dosages of CDDP and CA4P were fixed at 3.0 mg kg−1 and 30.0 mg kg−1 for in vivo studies. According to Fig. 5A, the tumor growth of the drug containing agent treated groups (G3 to G6) was obviously inhibited compared with the control groups (G1 and G2) at day 14 when the initial tumor treatment volume was around 110 mm3. The antitumor efficacy of the drug co-loaded gel (G6) showed an obvious advance compared with those of G3 and G5. However, the tumor inhibition rates of G3–G5 were also significantly improved compared with those of the control groups (G1 and G2). In contrast, when the initial treatment tumor volume was increased to 370 mm3, the tumor growth treated with the single drug loaded gel or free drugs showed no obvious suppression compared to the control groups at day 12. However, the group treated with the CA4P and CDDP co-loaded gel still exhibited an obvious tumor inhibition efficacy compared with other experimental groups (Fig. 5C).
image file: c8bm01442f-f5.tif
Fig. 5 The evaluation of antitumor efficacy with different initial tumor treating volumes in vivo. (A, C) Average tumor volumes and (B, D) average body weights with the single treatment of various therapeutics (G1, saline; G2, blank gel; G3, CA4P@Gel; G4, CDDP@Gel; G5, free CA4P and CDDP; G6, CA4P&CDDP@Gel) when the tumor volumes reached ∼110 mm3 (A, B) and ∼370 mm3 (C, D). (CDDP dosage: CDDP 3.0 mg kg−1; CA4P dosage: 30.0 mg kg−1) (n = 6, values were analyzed by one-way ANOVA for A and C, data depict mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001).

Furthermore, the therapeutic efficacy of the drug co-loaded gel was also evaluated by pathology examination. As displayed in Fig. 6, H&E staining images indicate that all the drug containing agent treated groups (G3–G6) showed obvious cellular apoptosis compared to both the saline and blank gel treated ones (G1 and G2). Additionally, the drug co-loaded gel treated group presented the highest extent of cellular apoptosis among all groups.22 Similar results were also confirmed by the TUNEL assay. There was almost no obvious red fluorescence signal in the saline and blank gel treated groups (G1 and G2). Nevertheless, the red fluorescence with different intensities was observed from the drug containing agent treated group (G3–G6). It is noteworthy that, the group treated with the CA4P and CDDP co-loaded gel (G6) exhibited the highest fluorescence intensity among the treated groups. Moreover, the body weight showed no obvious loss during the treatments (Fig. 5B and D). Collectively, these results demonstrated that this drug co-delivery strategy had the ability to manage the growth of the relatively bulky tumors.

image file: c8bm01442f-f6.tif
Fig. 6 Ex vivo H&E staining and TUNEL assay of C26 tumor tissues with the single treatment of various therapeutics (G1, saline; G2, blank gel; G3, CA4P@Gel; G4, CDDP@Gel; G5, free CA4P and CDDP; G6, CA4P&CDDP@Gel) when the tumor volumes reached ∼370 mm3 (scale bar: 100 μm).

4. Conclusions

In this work, we have developed a new strategy for enhanced local colon cancer (C26) treatment based on the CA4P and CDDP co-loaded mPEG-b-PELG thermo-gelling hydrogel. The mPEG-b-PELG hydrogel exhibited good biocompatibility and tunable biodegradability, which guarantee its flexible biomedical applications. Furthermore, the group treated with the CA4P-loaded gel exhibited enhanced intratumoral hemorrhaging behavior compared with the group treated with free CA4P within 48 h. The in vivo antitumor results suggested that the CA4P and CDDP co-loaded gel depot showed the highest antitumor efficacy in the C26 bearing mice with a single peritumoral injection. This drug co-delivery strategy based on the thermo-gelling polypeptide hydrogel holds potential for enhancing localized antitumor therapy.47,48

Conflicts of interest

There are no conflicts to declare.


We are grateful to the financial support from the National Key Research and Development Program of China (2016YFC1100701), the National Natural Science Foundation of China (projects 51773199, 51622307, and 51833010), the Science Innovation Projects of the Changchun University of Science and Technology (XJJLG-2016-08) and the start-up package at the UCLA.

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Electronic supplementary information (ESI) available. See DOI: 10.1039/c8bm01442f

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