Dong Wan, Sunfan Li, Jianxin Zhang, Guilei Ma*, Jie Pan,*

1 School of Chemistry and Chemical Engineering, Tiangong University, Tianjin 300387, China

2 The Tianjin Key Laboratory of Biomaterials, Institute of Biomedical Engineering, Peking Union Medical College & Chinese Academy of Medical Sciences, Tianjin 300192, China

Keywords:Micelles Drug delivery Tumor Enzyme Polymers Peptide

A B S T R A C T Compared with physical drug-loaded nanocarriers, polymeric prodrug micelles have many advantages such as high drug loading and enhanced stability in blood,so they have great potential in cancer therapy.However,these micelles have a big disadvantage,which cannot achieve long-term circulation in vivo and high absorption of tumor cells simultaneously, resulting in low administration efficiency and poor therapeutic effect on cancer. To solve problems of traditional polymeric prodrug micelles, novel polymeric micelles with tumor microenvironment response were designed in this work. The prodrug formed by covalently linking D-α-tocopherol polyethylene glycol succinate (TPGS3350), peptide (Pep), and doxorubicin (DOX) (TPGS3350-Pep-DOX) was self-assembled into micelles by encapsulating DOX physically.When the micelles entered the tumor tissue, the long-chain polyethylene glycol (PEG) was sensitively cut by the matrix metalloproteinase 2/9(MMP2/9)enzyme,exposing the targeting molecule folate,then it entered the cell through the endocytic pathway mediated by the folate receptor.The drug loading content, encapsulation efficiency, critical micelle concentration, and invitro release of the micelles invented in this study were measured to characterize their properties. The particle size and zeta potential of micelles were characterized by dynamic light scattering. Images were scanned by transmission electron microscopes. In vitro cytotoxicity, cellular uptake, and in vivo antitumor effect evaluation experiments were measured to show that smart micelles have made much progress in material chemistry and drug delivery, making it possible to apply a stimulus-response carrier drug delivery system in clinical application.

1. Introduction

With its rapidly growing morbidity and mortality, cancer has become a top disease in the world [1]. Doxorubicin (DOX) widely applied in cancer chemotherapy presents various shortcomings such as poor water solubility,high toxic side effects,short circulation time in blood,reducing the effectiveness of the drug[2-4].To overcome these disadvantages, scientists have resorted to polymeric prodrug micelles,which were prepared by conjugating drugs with polymeric matrix to form prodrug,followed by self-assembly to fabricate micelles [5]. Compared to the free drug, prodrug micelles could effectively improve the drug’s water solubility,prolong its systemic circulation time, and reduce its toxic side effects on normal tissues [6-14].

Although widely applied for drug delivery systems, prodrug micelles still faced the major challenge of not being able to achieve long circulation in the blood and high uptake by tumor cells at the same time, which reduced the efficacy of cancer chemotherapy[6,10,15].Literature published in Nature Nanotechnology depicted that after entering the bloodstream, the nanocarriers were coated by proteins within 30 s, triggering the phagocytosis of mononuclear phagocytes,which led to their accumulation and rapid clearance in reticuloendothelial systems such as the liver and spleen[16].To overcome this challenge,D-α-tocopherol polyethylene glycol succinate (TPGS) as pharmaceutical excipients. TPGS3350,formed by the esterification reaction of PEG (polyethylene glycol)3350 and vitamin E succinate, had an amphiphilic structure. It has been found that TPGS could enhance the solubility of various drugs,prolong blood circulation time,improve cellular absorption,and increase the cytotoxicity of drugs[17,18].At present,TPGS has been widely used in various drug delivery systems [19-21]. The micelle with PEG can weaken the binding of micelles to proteins in the blood,thus prolonging the circulation of micelles. However,once nanoparticles reach the tumor site, PEG on its surface will inhibit the recognition and absorption of micelles by tumor cells,resulting in low cancer treatment efficiency [22-26]. Therefore, it is urgent to design a new generation of prodrug micelles to achieve long circulation in the blood and high absorption of tumor cells.

Currently, many studies focused on the nanocarriers in response to the tumor microenvironment, which improved anticancer efficacy and reduced side effects on normal tissues [2,26-29]. Matrix metalloproteinase (MMP) is closely related to tumor invasion and metastasis, and it has become one of the hot spots in the field of anti-cancer [30]. Besides, MMP-responsive nanocarriers have been widely exploited in the field of drug delivery [31-36]. In our previous study, the MMP2/9-responsive nanocarriers carrying paclitaxel were designed and characterized its surface morphology, drug loading, drug release in vitro [37]. Moreover, a series of in vitro and in vivo experiments were carried out to explore its high efficiency in drug delivery and anti-cancer capabilities. The results confirmed that these nanocarriers achieved the long circulation in the blood and high uptake by tumor cells at the same time, and then improved the therapeutic effect and reduced the toxic and side effects of nanocarriers. Nevertheless,they still demonstrate various deficiencies such as low drug loading and instability in blood. Therefore, it is desirable to design novel enzyme-responsive micelles to improve the treating efficacy of cancer and diminish their toxic side effects on normal tissues.

Herein, MMP2/9-responsive micelles (DOX-loaded TPGS3350-Pep-DOX /TPGS-Folate micelles, denoted as TPD&TF) were developed to overcome the limitations of traditional micelles. DOX was encapsulated in the core of micelles by π-π stacking between free DOX and DOX in TPGS3350-Pep-DOX prodrug,which could not only increase the drug loading of micelles but also stabilize micelles by reducing the critical aggregation concentration, maintain their structure and enhance their stability in the process of blood circulation. We have introduced peptide technology into the preparation of polymeric prodrugs, taking advantage of the special conditions of the tumor microenvironment to modify the morphology of micelles to make them more readily available to tumor cells.The micelles designed in this work had the advantages of long circulation in vivo and high cellular uptake, all of which greatly improved the efficiency of cancer chemotherapy and reduced its toxic and side effects on healthy tissues and organs(Fig. 1).

Fig. 1. The preparation of TPD&TF micelles and drug delivery in vivo.

Fig. 2. The synthetic route of TPGS3350-Pep-DOX prodrug.

Fig. 3. 1H NMR spectrum of TPGS3350-Pep-DOX prodrug.

Fig. 4. (a) TEM image of TPD&TF; (b) The size of micelles incubated with or without MMP2/9.

Fig. 5. In vitro drug release profile of micelles.

2. Materials and Methods

2.1. Materials

N-hydroxysuccinimide (NHS),N,N′-dicyclohexylcarbodiimide(DCC),N,N-dimethylformamide (DMF), Triethylamine (TEA),dimethyl sulfoxide (DMSO), Folic acid, glutaric acid, dichloromethane (DCM) was supplied from Sinopharm Chemical Reagent Co.Ltd.Next,DMF was purified by dehydration over 4A molecular sieves at room temperature. NH2-PEG-NH2(MW: 3350 Da) was obtained from Beijing Kaizheng Joint Medical Technology Co. Ltd.D-α-Tocopherol Succinate (alpha-TOS) was from Aladdin (China).4′,6-diamidino-2-phenylindole (DAPI) and Cell counting kit-8 assay(CCK-8)were purchased from Beyotime Institute of Biotechnology, China. DOX was purchased from Beijing Huafeng Lianbo Technology Co. Ltd, China. Phosphate buffered saline (PBS), fetal bovine serum (FBS), and RPMI 1640 medium were all supplied from the Beijing HyClone. MMP2/9 (Collagenase, type 4) was purchased from CHI Scientific in China. All other chemicals were obtained from J&K Chemicals.A murine melanoma cancer cell line(B16) was provided from the cell culture center of the Institute of Basic Medical Sciences(Chinese Academy of Medical Sciences).All the experiments were studied on mice following the Guiding Principles for the Care and Use of Laboratory Animals, Peking Union Medical College, China.

2.2. Polymer synthesis

2.2.1. Synthesis of Pep

The MMP2/9-cleavable Pep (PVGLIG) was synthesized through the standard Fmoc protected solid-phase peptide synthesis (SPPS)method [38]. After deprotection, the polypeptide was cleaved by trifluoroacetic acid and purified by precipitation with excess anhydrous ether.

2.2.2. Synthesis of TPGS -Folate copolymers

The synthetic method of TPGS-Folate was described in previous studies of our group [37,39,40]. In brief, Folate was first activated with DCC and NHS, then reacted with TPGS (molar ratio 2:1) at room temperature for 24 h. Next, the mixture was precipitated in cold diethyl ether, dialyzed against deionized water for 48 h, and freeze-dried to gain TPGS-Folate.

2.2.3. Synthesis and characterization of TPGS3350-Pep-DOX prodrug

TPGS3350-COOH was first synthesized through exploiting α-TOS,PEG3350, DCC, NHS, and glutaric acid as reported in our previous study [37,40]. Next, TPGS3350-COOH was coupled with MMP2/9-cleavable PepviaNHS to form TPGS3350-Pep. In detail, TPGS3350-COOH(1 mmol)was activated in DMF(5 ml)with DCC(1.2 mmol)and NHS (1.2 mmol) at room temperature for 6 h. Pep (1.2 mmol)were further added into the above solution to stir for 24 h at room temperature to obtain TPGS3350-Pep.Additionally,DOX(1.2 mmol),TEA (6 mmol), TPGS3350-Pep (1 mmol), DCC (1.5 mmol) and NHS(1.5 mmol) in DMF were reacted for 24 h at 37 °C. After the reaction, the crude product was purified by dialysis against deionized water for 48 h (MWCO 3350 Da) to remove unreacted impurities.The final product of TPGS3350-Pep-DOX was obtained through freeze-dried. TPGS3350-DOX prodrug was also synthesized similarly.

TPGS3350-Pep-DOX dissolved in DMSO-d6was confirmed by nuclear magnetic resonance spectrometer (1H NMR) at 300 MHz(Bruker AVANCE AV 300,Germany). Gel permeation chromatography (GPC, Waters-2410 system) was utilized to determine the molecular weight of the prodrug.

2.3. Fabrication and characterization of TPD& TF

TPD&TF micelles were fabricated by thin-film hydration[36].In brief, the TPGS3350-Pep-DOX (10 mmol) was dissolved in a roundbottomed flask with 2 ml DCM together with TPGS-Folate(10 mmol) and DOX (1 mmol). After being fully dissolved, DCM was removed by rotary evaporation, a thin film was formed on the bottom and the thin film was dissolved in 3 ml of deionized water to obtain TPD&TF. Finally, after filtered through a 0.45 μm sterile filter, the final product was obtainedviafreeze-drying.DOX-loaded TPGS3350-DOX/TPGS-Folate micelles (TD&TF) were also fabricated with a similar procedure.

The critical micelle concentration(CMC)was determined by fluorescence probe techniques with 1,6-diphenyl-1,3,5-hexatriene(DPH) as a fluorescence probe through the Fluorescence spectrophotometer (F-280, Gangdong, and Tianjin, China) [41-44].The particle size, size distribution, and zeta potential of micelles were analyzed with dynamic light scattering(DLS,Nano-ZS90 Zeta Size, and Malvern, UK). The surface morphology of micelles was observed with transmission electron microscopy (TEM, Tecnai,USA).

2.4. The cleavage of micelles by MMP2/9

The cleavage of TPD&TF and TD&TF by MMP2/9 was investigated through enzymatic hydrolysis. Micelles were cultured with or without collagenase (type 4) at 25 °C for 24 h. The change in hydrodynamic diameters and morphology of micelles were recorded with DLS and TEM respectively.

2.5. Drug loading content and encapsulation efficiency

Drug loading content and drug encapsulation efficiency of micelles were evaluated with the Fluorescence spectrophotometer(F-280,Gangdong,Tianjin,China)at 586 nm emissions and 480 nm excitation.The drug loading content(DLC)and drug encapsulation efficiency (DEE) are calculated using the following equations:

2.6. In vitro drug release

The in vitro drug release of the micelles was evaluated by dialysis [45]. Briefly, 10 mg micelles were dissolved in 5 ml PBS containing collagenase(type 4)and dialyzed in 30 ml PBS.Remove the release medium (3 ml) at regular intervals, and the released amount of DOX from micelles was analyzed with Fluorescence spectrophotometer (F-280, Gangdong, Tianjin, China) under 480 nm excitation.

2.7. In vitro cytotoxicity

In vitro cytotoxicity of the micelles was measured withCCK-8 assay.B16 melanoma cells were seeded in a 96-well plate containing 200 μl medium at the density of 5000 cells per well. After culturing for about 12 h, the primary medium was replaced with a drug medium to make the DOX equivalent concentration of 0.5-1 5 μg·ml-1. After a while, cells were washed with PBS and treated with CCK-8 assay for 2 h,the absorbance of each well was recorded at 450 nm with a microplate reader(Perkin-Elmer).The cell viability (CV) was calculated by the following equation:

2.8. In vitro cellular uptake

In vitro cellular uptake of micelles by B16 cells was observed by the confocal laser scanning microscope (CLSM, Zeiss LSM710,Germany). B16 cells (1 × 104cells) were cultured in the medium for 24 h. Then the cells were cultured in the medium containing 0.125 mg·ml-1TPD & TF and TD & TF for 2 h and 4 h respectively,and the culture medium was taken out and the cells were washed with PBS.Next, the cells were fixed with 4%paraformaldehyde for 20 min, and the nuclei were stained with DAPI.

2.9. In vivo antitumor effect evaluation

B16 melanoma cells (100 μl) suspended of PBS were injected subcutaneously into the right-back of female BALB/c mice (about 6 weeks old).When the tumor volume reached 50 mm3,mice were injected intravenously by PBS, TPD&TF, and TD&TF (5 mg·kg-1of DOX equivalently) every third day. The tumor volume and the bodyweight of mice were recorded every three days.Tumor volume ＝

3. Results and Discussion

3.1. Synthesis and characterization of TPGS3350-Pep-DOX

The detailed synthetic route of TPGS3350-Pep-DOX is illustrated in Fig. 2. Firstly, MMP2/9 cleavable Pep was synthesized. Next,TPGS3350-Pep was fabricated by linking TPGS3350with PVGLIG and then react with DOX. Moreover, MMP2/9 non-responsive TPGS3350-DOX was prepared as a control.

Fig. 6. The cell viability of B16 cells incubated with TPD&TF, TD&TF and DOX for 24 h (a), 48 h (b) and 72 h (c).

It can be seen from Fig. 3 that the peaks at δ = 3.41-3.57 displayed the characteristic peaks of -CH2-CH2CH2O-. The characteristic peaks of hexapeptide were found at δ = 1.04-1.25(-CH3), δ = 5.88-6.03 (-CO-NH-), and δ = 1.89-1.91 (-CH2-).In addition, the characteristic peaks at δ = 0.81-0.85 were attributed to α-TOS in TPGS3350. The peaks of benzene in DOX were located at δ = 7.87-8.02. Therefore, it can be verified that the TPGS3350-Pep-DOX prodrug was synthesized successfully according to the results of1H NMR.

Fig. 7. CLSM images of B16 cells incubation with micelles for 2 h and 4 h.

3.2. Characterization of the micelles

As shown in Table 1, the hydrodynamic diameter was about 151 nm,the zeta potential of TPD&TF micelles was-5.65 mV,indicating the stability of its suspension. The spherical morphology of TPD&TF micelles observed with TEM had a mean diameter of 137 nm (Fig. 4(a)), which was small compared with DLS. Studies have shown that particles with an average particle size below 200 nm and a weak negative zeta potential could escape from the reticuloendothelial system (RES) to achieve long-distance circulation in the blood, and promote the accumulation of particles in the tumor by enhancing the permeability and retention (EPR)effect [46,47].

Table 1 The drug loading content, drug encapsulation efficiency, particle size and zeta potential of micelles

DLCDEE are important indicators to evaluate drug performance.To increase the content of DOX in micelles, free DOX and conjugated DOX in prodrug were encapsulated in the core of them by π-π stacking. Furthermore, the DLC and DEE of TPD&TF micelles were 15.15% and 60.25%. At the same time, it can be found in Table.1 that the DLC and DEE values of TPD&TF and TD/TF are almost similar, indicating that the peptides in the micelles have no significant effects on the DLC and DEE of the micelles.Additionally, the critical micelle concentration (CMC) of TPD&TF determined with the DPH probe was approximately 0.045 mg·ml-1,which implies stable micelles could be formed at low material concentrations.The low value of CMC is beneficial to improve the stability of micelles during an intravenous injection [48].

3.3. Cleavage of micelles by MMP2/9

The response characteristics of micelles to MMP2/9 were tested by adding collagenase (type 4) to simulate the extracellular microenvironment of the tumor site. It can be seen from Fig. 4(b)that two distinct peaks were observed in the DLS curve of TPD &TF micelles 24 h after MMP2/9 was added,indicating that the average sizes of these micelles were about 30 nm and 230 nm,respectively.This might because the peptides in TPD&TF were cut to form new micelles and TPGS3350separated from micelles and aggregated.

Fig. 8. (a) The tumor volume and (b) body mass of mice treated with saline,TPD&TF, TD&TF and free DOX, and (c) the tumor images with saline, TPD&TF micelles, TD&TF and free DOX after 21 d treatment.

3.4. In vitro DOX release from micelles

Fig. 5 illustrates the cumulativein vitrorelease of TPD&TF and TD&TF with or without MMP2/9 enzyme in 70 h in PBS(pH=7.4).Approximately 59.78%of DOX in TPD&TF micelles incubated with MMP2/9 was released for 70 h.In contrast,only 27.13%of DOX in TD&TF micelles in MMP2/9 was released after 70 h,which further confirmed the enzyme-responsive characteristics of TPD&TF micelles.Furthermore,there was no obvious difference in cumulative DOX release between TPD&TF and TD&TF without MMP2/9. Overall, these findings implied that TPD&TF micelles possess the MMP2/9-responsive property,which can achieve rapid release of DOX in cancer cells under the action of MMP2/9.

3.5. In vitro cytotoxicity and cellular uptake

The CCK-8 assay was utilized to evaluate thein vitrocytotoxicity of micelles on B16 murine melanoma cancer cells at 24 h,48 h,and 72 h. With a glance at Fig. 6, at the same DOX concentration,there was high toxicity of TPD&TF to B16 cells for 24 h, 48 h, and 72 h in comparison with free DOX and TD&TF.For instance,the cell viability of TPD&TF was 21.0% while TD&TF was 30.6% and free DOX was 24.3% at a drug concentration of 15 μg·ml-1after 24 h.Moreover, it is clear to see TPD&TF demonstrated higher cytotoxicity for B16 cells than the free DOX at all different drug concentrations, the result suggested the therapeutic efficacy of it was remarkable. In addition, the cytotoxicity of all formulations increased with incubation time. Thus, we can conclude that the sensitivity of TPD&TF to MMP2/9 is beneficial to increase the uptake of micelles by tumor cells.This phenomenon may be attributed to active targeting caused by the exposure of folate after the PEG layer falls off the surface of the nanoparticles under the action of MMP2/9.

In vitrocellular uptake was further investigated with CLSM.Compared with TPD&TF (Fig. 7B1) after 2 h of incubation, TD&TF in B16 cells (Fig. 7A1) showed weak red fluorescence around the blue nucleus of DOX, indicating that B16 cells internalized more TPD&TF. Moreover, after 4 h incubation, similar cellular internalization behavior was noticed in Fig. 7C1 and 7D1. These findings suggested that TPD&TF demonstrated higher intracellular accumulation than TD&TF after 2 h and 4 h incubation with B16 cells.Consequently,TPGS3350fell off the surface of TPD&TF under the action of MMP2/9 at the tumor site and presented the targeting ligand of folate, which led to increased uptake of micelles by B16 cells. The elevated cellular uptake of TPD&TF in Fig. 7 is consistent with the findings fromin vitrocytotoxicity in Fig. 6.

3.6. In vivo antitumor effect

A B16 melanoma xenograft model was established to verify the anti-tumor efficacy of micelles. Fig. 8(a) demonstrated the change of tumor volume in mice with different formulations every 3 d.

As indicated in Fig.8(a),the tumors of mice injected with saline grew rapidly within 21 d, and their volume increased 4.2 times compared with the initial tumor. After 21 d, TD & TF and free DOX showed a 2.5-fold and 3.3-fold growth of the tumor volume respectively, while TPD&TF expressed high tumor inhibition with only 1.9-fold, which was about 46.3% of the normal saline group.Meanwhile, the body mass of mice was monitored to determine the toxic side effects of formulations.

It is demonstrated in Fig. 8(b) that the body mass of DOXtreated mice decreased markedly after 21 d, indicating that DOX has serious toxicity to mice. Notably, there was no significant loss of body mass in TPD&TF and TD&TF after 21 d(Fig.8(b)),meaning that these formulations exhibited good biocompatibility. Furthermore,it can be observed in Fig. 8(c)that the tumor images of representative mice treated with various formulations collected after 21 d,which denoted TPD&TF have the best tumor inhibition effect compared with other groups.

4. Conclusions

In summary, TPD&TF micelles were developed in this work. In comparison to previous anti-cancer agents, this kind of novel micelle offers many advantages such as high drug loading, active targeting,and detachment of TPGS3350from the tumor site to promote efficient drug delivery,resulting in excellent anti-tumor efficiency and low side effects to normal tissues. In a word, TPD&TF designed in this work with multi-functional properties will be a promising platform for the clinical treatment of cancer.

Acknowledgements

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors are grateful for financial support from the National Natural Science Foundation of China (22078246, 81673027), Tianjin Natural Science Fund for Distinguished Young Scholars(17JCJQJC46400), CAMS Innovation Fund for Medical Sciences(CAMS-I2M-3-026).