Reduction-responsive modification-induced higher efficiency for attenuation of tumor metastasis of low molecular weight heparin functionalized liposomes

Abstract

For oncotherapy, a delivery system that can effectively prevent and attenuate tumor metastasis is greatly desirable. Herein, we report a novel delivery system of liposomal doxorubicin with a reduction-responsive modification of low molecular weight heparin (LMWH) to realize higher efficacy in the prevention of tumor metastasis. A series of DOX liposomal formulations of crude DOX-Lip, reduction-insensitive LMWH-DOX-Lip and reduction-responsive LMWH-ss-DOX-Lip were prepared. The reduction-responsive modification of DOX liposomes with LMWH chains resulted in higher cellular uptake, higher cytotoxicity of DOX and higher anti-invasion and anti-migration abilities of LMWH compared with reduction-insensitive liposomes, owing to the fast release of DOX and LMWH in the tumor cytoplasm. Meanwhile, the evaluation of heparanase expression was also adopted to reveal the possible underlying mechanism of the effects of LMWH on the attenuation of tumor metastasis. In vivo pulmonary melanoma metastasis assays confirmed the enhanced drug efficacy and lower side effects of LMWH-ss-DOX-Lip compared with crude DOX-Lip or the reduction-insensitive control group. These results may be attributed to the rational design of the drug carriers with a synergistic combination of LMWH moieties and DOX loaded liposomes via reduction-responsive modification.

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1. Introduction

Tumor metastasis is the greatest threat in cancer treatment and is the leading cause of human mortality after surgery resection of tumor tissues.¹ Currently, much attention is being paid to overcoming this issue; delivery systems that can effectively attenuate tumor metastasis are strongly needed.^2–4

Currently, chemotherapy is a conventional, useful method for the prevention of tumor metastasis by killing remaining tumor cells, and several cycles of chemotherapy after surgery can be regarded as a standard cure for cancer patients.⁵ However, efficiency usually correlates with severe adverse effects, and it is therefore necessary to balance chemotherapy efficiency with the patient's quality of life.⁶ Thus, the key point is to find a feasible way to realize enhanced anti-tumor metastasis abilities and reduce possible side effects.

Metastasis comprises a succession of steps, from the infiltration of cancer cells into blood and survival in the blood circulation, to the infiltration of foreign organs.⁷ We believe that each step could serve as a target and inhibitors of each step could function as synergistic treatments with conventional chemotherapy.

Low molecular weight heparin is the enzymolytic derivative of heparin, a natural sulfated glycosaminoglycan. Both heparin and LMWH were first used in the treatment of cancer-associated thrombosis in the clinic.^8,9 Fortunately, it has been found that patients can survive longer with lower rates of tumor metastasis if treated with heparin or LMWH.^10,11 Even though the mechanisms are believed to be complicated and still remain unclear, much meaningful work has already proceeded with heparin and LMWH to prevent tumor metastasis both in preclinical and clinical trials.^12–14 However, LMWH itself has limited activity against the inhibition of tumor growth;¹⁵ it can exhibit a synergistic effect for the prevention and treatment of tumor metastasis through co-delivery with other anti-tumor drugs via proper delivery systems.

Doxorubicin (DOX) is an anthracycline antibiotic that has a broad anti-tumor spectrum. Previous reports have demonstrated that intravenous delivery of free DOX alone usually results in fast clearance and severe toxicity of cardiotoxicity and myelosuppression, etc.¹⁶ Therefore, various new functional delivery systems have been developed.^17–20 Among these, DOX loaded liposomes have undoubtedly achieved the greatest success. For example, nonpegylated liposomal doxorubicin of Myocet® and pegylated liposomal doxorubicin of Doxil® are both approved by the US Food and Drug Administration and have been used in the clinic for years.^21,22 In addition to behaving as an effective formulation in curing primary cancers, liposomal DOX was also widely used in the treatment of recurring tumors, such as metastatic breast cancer or recurrent ovarian cancer.²³ However, there is still great potential for improvement by addressing the remaining problems of high progression and mortality level.²¹

Our recent study illustrates the feasibility of the reduction-insensitive conjugation of LMWH with doxorubicin liposomes via charge interaction; we achieved positive results.²⁴ However, during our study, we found that direct insensitive conjugation led to lower cytotoxicity of DOX; the anti-tumor metastasis potential of LMWH was also reduced, which may decrease the overall performance to some extent. Thus, a more efficient drug carrier design was necessary.

In recent years, environmentally sensitive drug delivery vehicles have demonstrated increasing advantages. Among these, reduction-responsive drug delivery systems are capable of fast release of loaded drugs in the tumor cytoplasm owing to the high level of the internal biological reduction stimulus of glutathione (GSH);^25–33 this enabled the vehicles to be stable and have low clearance during blood circulation and fast release once entrapped into tumor cells, enhancing the drug efficacy.

Thus, in this work, reduction-responsive modification was employed to design and synthesize LMWH functionalized DOX liposomes to realize synergistic anti-tumor and anti-tumor metastasis effects, as illustrated in Fig. 1. The present paper aims to report the rational design of this system and the experimental results for the first time.

Fig. 1 (A) Chemical structure of DOX and LMWH, and they are co-delivered via the vehicle of liposome. (B) Firstly, the LMWH modified redox-responsive DOX liposome (LMWH-ss-DOX-Lip) underwent receptor mediated endocytosis. Secondly, owing to the reduction environment of GSH in tumor cells, the disulfide linkage of LMWH-ss-DOX-Lip breaks, leading to the faster release of DOX and LMWH in tumor cytoplasm. Finally, the released DOX resulted in cell apoptosis and released LMWH inhibited cell invasion and migration, exhibiting a synergistic effect on anti-tumor metastasis.

2. Materials and methods

2.1 Materials

Soybean phosphatidylcholine (SPC) was purchased from German Lipoid Corporation, and cholesterol (Chol) was obtained from Sigma-Aldrich Corporation. Low molecular weight heparin (LMWH, MW ∼ 4500 Da) was provided by Hebei Changshan Biochemical Pharmaceutical Co. Ltd. Doxorubicin hydrochloride (DOX·HCl) was obtained from Zhejiang Hisun Pharmaceutical Co. Ltd. 1-Ethyl-3-3-dimethylaminopropyl carbodiimide hydrochloride (EDC·HCl), N-hydroxysulfosuccinimide (sulfo-NHS), dithiothreitol (DTT) and stearylamine (SA) were purchased from Aladdin Reagent Co. Ltd. 3,3′-Dithioldipropionic acid di(N-hydroxysuccinimide ester) (DTSP) was bought from Heowns Biochem Technologies. All other chemicals and reagents were of analytical grade and were used without further purification.

2.2 Cell culture and animals

B16F10 cells (a murine melanoma cell line) were provided by KeyGEN BioTECH Co. Ltd. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, Gibco), 80 U mL⁻¹ penicillin and 0.08 mg mL⁻¹ streptomycin in 5% CO₂ atmosphere at 37 °C.

C57BL/6 male mice (∼20 g, 6 weeks old) were purchased from the Academy of Military Medical Sciences of the Chinese People's Liberation Army. All animal experiments complied with the Guide for the Care and Use of Laboratory Animals of China Pharmaceutical University.

2.3 Preparation of liposomes

2.3.1 Preparation of crude liposomes. The crude liposomes were prepared by the film dispersion method. Briefly, the lipids (100 mg SPC, 10 mg Chol) were dissolved in chloroform and evaporated to form a thin film. 5 mL ammonium sulfate solution (300 mM) was added for hydration at 37 °C for 60 min. Then, the suspension was sonicated for 30 min and passed through a 0.22 μm filter. The liposomes were dialyzed against 400 mL PBS solution (pH 7.4) for 1.5 h twice to remove the unloaded ammonium sulfate.

For the preparation of DOX loaded liposomes, 2 mg mL⁻¹ DOX solution (in PBS medium) was added to the liposome solution with a drug-to-lipid ratio of 1/10 (mg/mg), and then incubated in 60 °C water bath for 30 min.

2.3.2 Preparation of LMWH modified liposomes.
2.3.2.1 Synthesis of LMWH disulfide conjugates (LMWH-DTSP-OSu). LMWH (180 mg, 0.04 mmol), EDC·HCl (22.4 mg, 0.12 mmol) and sulfo-NHS (9.0 mg, 0.04 mmol) were dissolved in 20 mL PBS (pH 7.2) and reacted for 30 min at room temperature for the activation of LMWH. To this mixture, 0.4 mmol ethylenediamine (24 mg) was added and the reaction was maintained with stirring for another 12 h. Then, the solution was dialyzed against distilled water for 48 h with a dialysis membrane (MWCO 3500), and the retentate was lyophilized to obtain a white solid product (LMWH-eda) with a yield of over 85%.

0.04 mmol LMWH-eda (180 mg) was dissolved in 3 mL pH 7.2 PBS; then, 3 mL DTSP/DMF solution (containing 48 mg DTSP) was added dropwise. After treatment with triethylamine (20 μL, 0.14 mmol), the mixture was reacted with stirring for 30 min at room temperature. The resulting solution was dialyzed against distilled water for 3 days to remove unreacted chemicals, and the retentate was lyophilized to obtain LMWH disulfide conjugate (LMWH-DTSP-OSu) with a yield of over 90%.

2.3.2.2 Modification of liposomes with LMWH. The preparation and drug loading of liposomes for redox-responsive LMWH-ss-liposomes (LMWH-ss-Lip) or reduction-insensitive LMWH-liposomes (LMWH-Lip) was similar to that of the crude liposomes described in 2.3.1, with the only difference being the lipid compositions of SPC, Chol and SA (SPC/CHOL/SA: 100/10/4 mg/mg/mg).

For the preparation of LMWH-ss-Lip, LMWH-DTSP-OSu (6.6 mg, 1.32 μmol) was dissolved in 3 mL PBS (pH 7.2), then mixed with the same volume of liposomes or DOX-liposome solutions (lipid concentration 10 mg mL⁻¹, lipid composition SPC/Chol/SA). After treatment with triethylamine (2 μL, 14 μmol), the mixture was stirred for 30 min at room temperature to obtain LMWH-ss-liposome or LMWH-ss-DOX-liposome.

For the preparation of LMWH-Lip, LMWH (0.004 mmol), EDC·HCl (0.012 mmol) and sulfo-NHS (0.004 mmol) were dissolved in 9 mL PBS (pH 7.2); after activation for 6 h at 4 °C, the same volume of liposome or DOX-liposome solution (lipid concentration 10 mg mL⁻¹) was added. The reaction progressed overnight to afford LMWH-Lip or LMWH-DOX-Lip.

2.4 Particle size, zeta potential, morphology and encapsulation efficiency

The liposome suspensions were diluted with PBS (pH 7.4). The particle size and zeta-potential were measured using a Zetasizer 3000 HAS. Each measurement was repeated three times.

The morphology of the liposomes was observed by transmission electron microscopy (TEM). Liposome suspensions containing 1% phosphotungstic acid were dropped onto a copper grid. After drying at room temperature, the copper grid was observed.

The encapsulation efficiency (EE) of drug loaded liposomes was evaluated through size exclusion chromatography (Sephadex G-50 column). 0.2 mL DOX loaded liposome solution was added to the top of the G-50 column with elution buffer of 0.2 mL PBS each time. After centrifugation at 1000 rpm for 3 min, the eluent was collected. The elution process was repeated three times and the UV absorption of the total eluents was measured at 480 nm after dilution with acidic isopropanol to a final volume of 10 mL, recorded as C_DOX,liposome. The total amount of DOX of the drug loaded liposome solution was measured directly without size exclusion chromatography. Its UV absorption was also measured at 480 nm after dilution with acidic isopropanol to the same volume of 10 mL, denoted as C_DOX,total.

EE (%) = C_DOX,liposome/C_DOX,total × 100% (1)

2.5 Toluidine blue assay of LMWH

The binding efficiency (BE) of LMWH was evaluated by the toluidine blue assay. Briefly, 0.2 mL LMWH-DOX-Lip or LMWH-ss-DOX-Lip suspension was added to an ultrafiltration tube (30 kDa) and centrifuged at 14 000 rpm for 60 min. The filtrate (containing free LMWH, not bound to the liposomes) was diluted with distilled water to 10 mL. A 2 mL sample was withdrawn and mixed with 3 mL toluidine blue solution (0.005% toluidine blue and 0.5% NaCl in 0.01 mol L⁻¹ HCl). The mixture was incubated in a water bath at 37 °C for 2 h. 3 mL of hexane was transferred to the mixture, which was stirred vigorously. The absorbance of the water phase was measured at 630 nm using UV-vis spectrophotometry, and the amount of free LMWH was calculated, denoted as LMWH_free. The total amount of LMWH in the liposome solutions was directly treated with toluidine blue without ultrafiltration, denoted as LMWH_total.

BE (%) = (LMWH_total − LMWH_free)/LMWH_total × 100% (2)

2.6 In vitro drug release

The in vitro release profiles of DOX from DOX-sol, DOX-Lip, LMWH-DOX-Lip, and LMWH-ss-DOX-Lip were investigated with the dialysis method. 1 mL sample solution (DOX 0.5 mg mL⁻¹) was sealed in a dialysis bag (MWCO 3500) and immersed in 50 mL PBS (pH 7.4) as the releasing buffer. The releasing conditions were 37 °C with a rotation speed of 100 rpm. At a predetermined time, 0.2 mL medium was taken out and replaced with an equal volume of fresh PBS. The released DOX was measured by HPLC with a mobile phase of H₂O (0.0288% SDS) : acetonitrile : methanol (50 : 50 : 6, v/v/v, pH 2.2), C18 column, 1 mL min⁻¹, 480 nm.

As a redox-responsive control, the release profile of LMWH-ss-DOX-Lip was also evaluated in the presence of 10 mM DTT.

2.7 In vitro cytotoxicity

B16F10 cells were seeded in a 96-well plate with a cell density of 6 × 10³ cells per well (drug incubation time 24 h) or 3 × 10³ cells per well (drug incubation time of 48 h). After 16 h, the culture medium was replaced by 200 μL medium containing a series of concentrations of DOX-sol, DOX-Lip, LMWH-DOX-Lip or LMWH-ss-DOX-Lip. After incubation for another 24 h or 48 h, the medium was withdrawn and 20 μL MTT solution (5 mg mL⁻¹ in PBS) was added. After incubating for 4 h at 37 °C, 150 μL DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was read by a microplate reader. The viability of the control group (untreated with liposomes) was regarded as 100%. The cell viability (%) was calculated as OD_{test group}/OD_{control group} × 100%.

2.8 In vitro cellular uptake evaluation

B16F10 cells were seeded in a 6-well plate with a cell density of 5 × 10⁵ cells per well. After incubation overnight in 1 mL RPMI 1640 medium (containing 10% FBS), the medium was replaced by 1 mL RPMI 1640 medium containing DOX, DOX-Lip, LMWH-DOX-Lip or LMWH-ss-DOX-Lip, with a final DOX concentration of 30 μg mL⁻¹. 3 hours later, the medium was removed and the cells were washed three times with cold PBS solution. After being fixed with immunol staining fix solution (30 min, room temperature) and stained with Hoechst 33258 (20 min, 37 °C), the cells were observed by laser confocal scanning microscopy (CLSM, Olympus), with excitation and emission wavelengths of 488 nm and 560 nm, respectively.

The in vitro cellular uptake was also semi-quantitatively analyzed by flow cytometry. Briefly, B16F10 cells were seeded in a 6-well plate with a cell density of 5 × 10⁵ cells per well. After incubating overnight, the medium was replaced by 1 mL RPMI 1640 medium containing DOX, DOX-Lip, LMWH-DOX-Lip or LMWH-ss-DOX-Lip, with a final DOX concentration of 30 μg mL⁻¹. After 0.5 h, 1 h and 3 h, the cells were washed with cold PBS. After dissociation with trypsin, the cells were resuspended in 1 mL PBS solution, followed by examination of DOX uptake by a flow cytometer, with excitation and emission wavelengths of 488 nm and 560 nm, respectively.

For the inhibitory competition control, the cells were incubated in medium containing 100 μg mL⁻¹ free LMWH for 0.5 h prior to treatment with LMWH-ss-DOX-Lip.

2.9 Effect of LMWH on cell invasion and migration

A Matrigel invasion chamber fitted with an 8 μm pore size membrane (Transwell chamber) was used to evaluate the effect of LMWH on cell invasion. After being diluted with equal amounts of culture medium (0% FBS), 30 μL Matrigel was coated on the Transwell chamber. Then, 500 μL culture medium (20% FBS) was added to the lower compartment as the attractant.

B16F10 cells were suspended in RPMI-1640 culture medium (0% FBS, 0.1% BSA) with a cell density of 5 × 10⁵ cells per mL, and 100 μL cells were seeded in the Transwell chamber. Saline, LMWH-sol, LMWH-Lip and LMWH-ss-Lip were added to the Transwell chamber with a final LMWH concentration of 100 μg mL⁻¹. After incubation for 24 h, the non-invading cells of the upper membrane surface were removed with a cotton ball. Then, the cells adhering to the lower side were stained with 0.1% crystal violet. The invasion cell number was counted in the same three fields on the diameter of all the groups.

The migration assay was similar to the invasion assay, although the transwell chamber was not coated with Matrigel.

2.10 RT-PCR analysis of the effect of LMWH on the gene expression of heparinase

The gene expression of heparinase was detected using real-time PCR to quantify the inhibition effect of LMWH on the expression of heparinase. B16F10 cells were seeded in a 24-well plate with a cell density of 1 × 10⁵ cells per well. After the cells adhering to the plates, the media were replaced by LMWH-sol, LMWH-Lip, and LMWH-ss-Lip solutions (LMWH 100 μg mL⁻¹, RPMI-1640 as the medium). For the control group, an equal amount of saline was added. After incubation for 24 h, the cells were trypsinized to extract the total cellular RNA using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA (2 μg) was reverse-transcribed into the first strand of complementary DNA (cDNA) with a Prime-Script® RT reagent kit (Thermo Fisher). The expression of heparinase was determined by real-time PCR (RT-PCR, Daan Gene DA7600) with a real-time PCR Master Mix kit (SYBR Green). Heparinase was amplified using its specific primers: sense primer 5-CGAT GTCT GTAG TGCT GGCT-3; antisense primer 5-TCTGATTGCTGCTGGATCTC-3. GAPDH, a housekeeping gene, was included as an endogenous reference using the following primers: sense primer 5-TAT GTC GTG GAG TCTA CTGGT-3; antisense primer 5-GAG TTG TCA TATTTCTCGTGG-3.

2.11 In vivo lung tumor metastasis assays

B16F10 cells were suspended in PBS with a cell density of 3 × 10⁶ cells per mL. Sixty C57BL/6 mice were randomly divided into 6 groups. For each group, free drug or liposome solutions were injected into the tail vein, with DOX doses of 5 mg kg⁻¹ for the DOX-sol, DOX-Lip, LMWH-DOX-Lip, and LMWH-ss-DOX-Lip groups and LMWH doses of 16 mg kg⁻¹ for the LMWH-sol group. 30 min later, 0.1 mL B16F10 cell suspension was inoculated through the tail vein. Subsequently, the mice were administered on day 4, 7, 10, 13, 16, and 19 after tumor cell inoculation with the same drug dose. On the 21^st day, the mice were sacrificed and their lungs were extracted. The anti-metastasis effect of the treatments was evaluated by the lung weight and the number of tumor colonies on the lung surface. The metastasis foci in the lung tissue were observed (×200) and counted (×40, 10 fields) by microscope.

2.12 In vivo pharmacokinetic evaluations

The pharmacokinetic study was carried out in SD rats (180 to 220 g). The rats were fasted one day before drug administration. DOX-sol, DOX-Lip, LMWH-DOX-Lip and LMWH-ss-DOX-Lip were administered to the rats via the tail vein at a DOX dose of 3.5 mg kg⁻¹. At predetermined times of 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h and 36 h after intravenous administration of different drug formulations, approximately 1 mL blood samples were withdrawn via the orbit. Plasma was obtained by immediate centrifugation of the blood samples at 5000 rpm for 10 min and was stored at −20 °C.

30 μL of an internal standard substance (alogliptin) was added to 270 μL plasma. The plasma was then deproteinized with 2 mL chloroform–methanol (4 : 1, v/v) solution. After vortexing for 5 min and centrifugation at 3000 rpm for 10 min, the chloroform layer was taken out and evaporated under a moderate stream of nitrogen gas. The residue was dissolved in 100 μL methanol, and DOX in the supernatant was analyzed with HPLC.

The HPLC conditions were as follows: Lichrospher C4 column (250 × 4.6 mm, 5 μm), mobile phase consisting of acetonitrile–methanol–0.2 mol L⁻¹ NaH₂PO₄ (pH 4.2) (20.5 : 20 : 59.5, v/v/v) at a flow rate of 1.0 mL min⁻¹, detection wavelength 254 nm, column temperature 30 °C. The retention times of DOX and the internal standard were approximately 13 and 5 min, respectively. The lower limit of quantitation value of DOX in rat plasma was 40 ng mL⁻¹.

2.13 Evaluation of bleeding time via cutting tail test

LMWH-sol, LMWH-DOX-Lip and LMWH-ss-DOX-Lip were administered to mice via the tail vein with LMWH doses of 6 mg kg⁻¹ or 16 mg kg⁻¹. One group was administered with saline as a control. After 30 min, the rats were anesthetized and a 5 mm incision from the tail tip was cut. The tail was placed vertically in 37 °C saline and the visible bleeding time was recorded as if there were no bleeding for 30 s.

2.14 Statistical analysis

Data were presented as mean ± SD. Statistical analysis was performed with the analysis of variance (SPSS16.0). p < 0.05 was considered statistically significant.

3. Results

3.1 Preparation of LMWH modified liposomes

The preparation scheme of LMWH modified liposomes is shown in Fig. 2. For the redox-responsible liposomes of LMWH-ss-liposome, the preparation approach consisted of three steps. First, the carboxyl group of LMWH reacted with ethanediamine (EDA) with the catalyst of EDC/NHS to construct an LMWH-amine derivative. Second, the amino-modified LMWH underwent an amidation reaction with DTSP to incorporate a disulfide bond. The synthesized conjugate of LMWH-DTSP-OSu (Fig. S1†) was characterized by ¹H-NMR (300 MHz, D₂O): δ = 2.72 (s, 4H, Su), δ = 2.92 (t, 4H, 2CH₂S). Finally, LMWH-DTSP-OSu reacted with the amino group of stearamine in the liposomes to yield LMWH modified redox-responsive liposomes (LMWH-ss-Lip). For the preparation of reduction insensitive liposomes (LMWH-Lip), the carboxyl group of LMWH directly reacted with the amino group of the liposomes with EDC/NHS as catalysts.

Fig. 2 (A) Schematic of synthesis of LMWH-DTSP-OSu. (B) Preparation scheme of LMWH-ss-DOX-Lip and LMWH-DOX-Lip.

3.2 Characterization of the liposomes

The particle sizes (PS) of the prepared blank liposomes or DOX-Lip were around 110 nm; after modification with LMWH, the PS slightly increased to about 120 nm (Table 1). According to references, a particle size within 20 to 200 nm is desirable for enrichment in tumor tissues based on the enhanced permeability and retention (EPR) effect. Thus, the prepared liposomes could meet this criterion. Meanwhile, the modification of LMWH could enhance the liposome stability in blood due to the high flexibility and hydrophilicity of the heparin molecules. The surface of the un-modified liposomes (lipid composition of SPC/Chol) was negatively charged, with a zeta potential of around −10 mV. For the modified liposomes, the zeta potential was about 20 mV for the protonation of the amino group of SA in the lipids (lipid composition of SPC/Chol/SA) (Table 1). The slightly positively charged surface could enhance the liposome stability during storage due to charge repulsion.

Table 1 The physiochemical characterization of prepared liposomes

	Particle size (nm)	Polydispersity index	Zeta potential (mV)	Entrapment efficiency (%)
Lip	111.1 ± 1.6	0.250 ± 0.009	−8.78 ± 0.58	—
DOX-Lip	111.9 ± 2.4	0.253 ± 0.019	−7.31 ± 1.27	97.5 ± 2.05
LMWH-DOX-Lip	117.0 ± 3.8	0.243 ± 0.030	17.28 ± 2.38	96.4 ± 2.10
LMWH-ss-DOX-Lip	123.3 ± 1.3	0.227 ± 0.010	23.21 ± 2.01	96.7 ± 2.20

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The toluidine blue assay was adopted to evaluate the binding efficiency (BE) of LMWH. As shown in Table S1,† the BE (%) was over 80%; this value decreased slightly as the LMWH feeding amount increased.

The morphology of the liposomes is shown in Fig. 3. The morphology of DOX-Lip was a sphere, while that of the LMWH-liposomes was sphere-like and fluffy, indicating the successful modification of LMWH molecules on the surface of the crude liposomes.

Fig. 3 The morphology of prepared liposomes of DOX-Lip (A), LMWH-DOX-Lip (B) and LMWH-ss-DOX-Lip (C). The scale bar represents 20 nm.

3.3 In vitro drug release

The in vitro DOX release profiles were evaluated by dialysis method. As shown in Fig. 4, as the control group, the drug release of DOX-sol was the fastest, with complete release within 6 hours. After encapsulation in the liposomes, the release rates decreased. For DOX-Lip, the cumulative release of DOX over 24 h was about 80%, while the releases were only 57% and 65% for LMWH-DOX-Lip and LMWH-ss-DOX-Lip (release medium without DTT), respectively.

Fig. 4 Release profiles of DOX out of the prepared liposomes (n = 3). Free DOX group (DOX-sol) was set as control.

In presence of 10 mM DTT, the DOX release rate was increased, with the cumulative release increasing to about 80% within 24 h, which is similar to that of DOX-Lip. The reason may be attributed to the reduction-induced breakage of the disulfide bond and the separation of the LMWH shield.

3.4 In vitro cellular uptake of drug loaded liposomes

The cellular uptake of DOX with different formulations was qualitatively observed by CLSM. As shown in Fig. 5A, the fluorescence intensity of DOX was strongest for the group of DOX-sol, due to the free drug entering into the cells more easily through passive diffusion. After DOX was loaded in the liposome (DOX-Lip), the fluorescence intensity became weaker; however, with the modification of LMWH, the cellular uptake of DOX was improved. Competitive inhibition tests were also performed. For cells pre-treated with 100 μg mL⁻¹ LMWH, the fluorescence intensity of LMWH-ss-DOX-Lip was similar to that of DOX-Lip, confirming the positive effect of LMWH on the cellular uptake of DOX loaded liposomes.

Fig. 5 (A) CLSM images of B16F10 cells after incubation with DOX loaded liposomes for 3 h. For each panel, the images from left to right present DOX fluorescence (red), Hoechst 33342 (blue, cell nucleus stained), bright field images and fluorescence merged images. (B) Flow cytometry analysis of B16F10 cells after incubation with DOX loaded liposomes for 0.5 h, 1 h and 3 h.

The cellular uptake of DOX with different formulations was also semi-quantitatively evaluated by flow cytometry, as illustrated in Fig. 4B. The cellular uptake was incubation time-dependent. As the incubation time increased, the mean fluorescence intensity (MFI) of the cells increased. Meanwhile, the cellular uptake abilities of all the tested groups were listed as DOX-sol > LMWH-ss-DOX-Lip > LMWH-DOX-Lip > LMWH-ss-DOX-Lip + LMWH > DOX-Lip, which is in agreement with the results of CLSM.

3.5 In vitro cytotoxicity of DOX loaded liposomes on B16F10 cells

The anti-proliferative effect of DOX loaded liposomes on tumor cells was evaluated by MTT assay. As shown in Fig. 6, the cytotoxicity of B16F10 cells was drug concentration dependent. DOX exhibited different half-maximal inhibitory concentration (IC₅₀) values in different formulations. Specifically, the values were 0.72 μg mL⁻¹ (DOX-sol), 6.20 μg mL⁻¹ (DOX-Lip), 0.95 μg mL⁻¹ (LMWH-DOX-Lip) and 0.82 μg mL⁻¹ (LMWH-ss-DOX-Lip), respectively, for 24 h incubation time. Afterwards, the IC₅₀ values of the different groups sharply decreased to 0.04 μg mL⁻¹ (DOX-sol), 0.35 μg mL⁻¹ (DOX-Lip), 0.28 μg mL⁻¹ (LMWH-DOX-Lip) and 0.10 μg mL⁻¹ (LMWH-ss-DOX-Lip), respectively, for 48 h incubation time. However, at a longer incubation time of 72 h, the IC₅₀ values did not decrease further compared with the value of 48 h (data not shown).

Fig. 6 In vitro inhibition rate of DOX in different groups against B16F10 cells at indicated incubation time (n = 3). Inhibition rate was calculated as 100% minus cell viability (%).

Even though DOX-sol presented the strongest anti-proliferative effect against B16F10 cells in vitro, the liposome formulations could exhibit better performance in vivo with higher bioavailability and lower adverse effects.

The cytotoxicity of the LMWH functionalized liposomes was greater than that of crude DOX-Lip, possibly due to the participation of LMWH in the transfer of drug loaded liposomes via specific peptide-mediated endocytosis. Also, the cytotoxicity of the redox-sensitive liposome (LMWH-ss-DOX-Lip) was higher than that of the reduction-insensitive liposome (LMWH-DOX-Lip) because the high GSH level in the tumor cells facilitated the disaggregation of LMWH, leading to fast release of DOX in the cytoplasm.

3.6 Effect of LMWH on cell invasion and migration

The Matrigel invasion assay is a classical approach to evaluate the invasion ability of tumor cells. As shown in Fig. 7, after treatment with free LMWH or LMWH modified liposomes, the invasion ability of tumor cells was significantly decreased compared with the control group (saline), illustrating the positive effects of LMWH on the inhibition of tumor metastasis. The invasion-inhibition ability order was LMWH-sol > LMWH-ss-Lip > LMWH-Lip > saline, indicating that the invasion inhibition ability of LMWH decreased after it was linked to the liposomes. Meanwhile, the disulfide bond of LMWH-ss-Lip enabled faster separation of LMWH, which may account for the relatively stronger invasion-inhibition ability of LMWH-ss-Lip compared with that of LMWH-Lip.

Fig. 7 Cell invasion analysis of B16F10 cells with Transwell assay after incubated with saline, LMWH-sol, LMWH-Lip and LMWH-ss-Lip for 24 h. The LMWH concentration was 100 μg mL⁻¹. (A) Typical images of invasion cells after stained with 0.1% crystal violet. Scale bar reads 30 μm. (B) Average invasion cell numbers in the same fields of denoted groups (n = 3). And t-test was calculated between the experimental groups and the control group (saline). Significant difference denoted as p < 0.05 (*), p < 0.01 (**).

The migration of B16F16 was also investigated by a Transwell chamber assay (without Matrigel coating). The results were similar to that of the cell invasion assay. As shown in Fig. 8, the presence of LMWH could significantly depress tumor cell migration.

Fig. 8 Cell migration analysis of B16F10 cells with Transwell assay (without Matrigel coating) after incubated with saline, LMWH-sol, LMWH-Lip and LMWH-ss-Lip for 24 h. (A) Typical images of migration cells after stained with 0.1% crystal violet. Scale bar reads 30 μm. (B) Average migration cell numbers in the same fields of denoted groups (n = 3). And t-test was calculated between the experimental groups and the control group (saline). *p < 0.05 and **p < 0.01.

3.7 Effect of LMWH on the expression of heparinase mRNA

The expression of heparinase is closely related to tumor metastasis. Heparinase is highly expressed in malignant tumors, while in benign tumours, heparinase expression is mild. Thus, efficient inhibition of heparinase expression is important for the attenuation of tumor metastasis.

Real-time PCR was adopted to detect the expression of heparinase mRNA in B16F10 cells after treatment with saline, LMWH-sol, LMWH-Lip and LMWH-ss-Lip. As shown in Fig. 9, the presence of LMWH significantly suppressed Hpa gene expression, with relative expressions of 49%, 89% and 80% for the LMWH-sol, LMWH-Lip and LMWH-ss-Lip groups, respectively, compared with the saline group.

Fig. 9 Relative Hap gene expression of B16F10 cells tested by QPCR after treated with saline, LMWH-sol, LMWH-Lip and LMWH-ss-Lip for 24 h. t-test was calculated between the experimental groups and the control group (saline). Significant difference denoted as p < 0.05 (*), p < 0.01 (**).

3.8 In vivo lung tumor metastasis assay

The mice pulmonary melanoma metastasis model was used to explore the anti-tumor metastasis ability of the different groups. The mean mass of the lungs after treatment with drug formulations is usually regarded as an indicator of tumor metastasis. The heavier the lung weight, the stronger the tumor metastasis. As shown in Fig. 10A, the mean lung mass of the LMWH-ss-DOX-Lip group was the smallest compared with the other groups, indicating that LMWH-ss-DOX-Lip has the strongest anti-tumor metastasis ability. Moreover, the extracted lung tissue was HE stained to evaluate the tumor metastasis nodes. As shown in Fig. 10B, for the saline group, the lung tissue was thoroughly infiltrated by tumor tissues, while for the LMWH-ss-DOX-Lip group, the tumor filtration was significantly attenuated. Tumor metastasis foci, another indication of the extent of tumor metastasis, were also counted according to the HE stained images of lung tissue, with the foci number order of LMWH-ss-DOX-Lip < LMWH-DOX-Lip < DOX-Lip < DOX-sol < LMWH sol < saline, as shown in Fig. 10C.

Fig. 10 In vivo lung tumor metastasis of B16F10 cells after a cycle of therapy with saline, LMWH-sol, DOX-sol, DOX-Lip, LMWH-DOX-Lip and LMWH-ss-DOX-Lip. (A) Mice lung mass of different groups at day 21 (n = 10). Student's t-test was calculated between saline group and other experimental groups, with significant difference of p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***); t-test was also calculated between LMWH-ss-DOX-Lip group and other experimental groups, with significant difference of p < 0.05 (#), p < 0.01 (##) and p < 0.001 (###). (B) HE-stained images of lung tissue at day 21. (C) Metastasis tumor foci accounted based on the HE-stained images of lung tissue. The symbol denotation of p values was same with that of Fig. 9A. (D) Body weights of different groups during the whole therapy (n = 10).

The body weights of the mice were recorded, as shown in Fig. 10D. For the DOX-sol group, the body weight of the mice significantly decreased to a value of only 16.4 g (12% decreased) at day 21, indicating severe toxicity during the therapy. For DOX-Lip, the body weight decreased about 5%. Meanwhile, for the other DOX loaded groups (LMWH-DOX-Lip and LMWH-ss-DOX-Lip), the body weights remained nearly constant during the therapy period, illustrating mild and slight toxicity. For the saline group, the body weight increased due to the much heavier lung mass resulting from significant tumor metastasis.

3.9 In vivo pharmacokinetic evaluations

The plasma concentration–time profiles of DOX after intravenous injection of DOX-sol, DOX-Lip and LMWH-ss-DOX-Lip at a single dose of DOX (3.5 mg kg⁻¹) are shown in Fig. 11. The plasma DOX concentrations were fitted with a single compartment model. Compared with DOX-sol, LMWH-DOX-Lip and LMWH-ss-DOX-Lip provided significantly elongated plasma t_1/2.

Fig. 11 Plasma concentrations–time curves of DOX in SD rats after intravenous injection of DOX-sol, DOX-Lip, LMWH-DOX-Lip and LMWH-ss-DOX-Lip with DOX dose of 3.5 mg kg⁻¹ (n = 3). The data were fitted with single compartment model with plasma t_1/2 denoted in the figure. Significant differences were exhibited between Dox-Lip and LMWH-DOX-Lip (p < 0.001, ###), Dox-Lip and LMWH-ss-DOX-Lip (p < 0.05, #).

3.10 Bleeding time via cutting tail test

LMWH has possible side effects of elongation of bleeding time. As shown in Fig. 12, after treatment with LMWH-sol, LMWH-DOX-Lip and LMWH-ss-DOX-Lip, the blood coagulation time was significantly prolonged and was found to be LMWH dose dependent. Fortunately, when LMWH was linked to liposomes, the adverse effects were weakened to some extent, as demonstrated by the shortened bleeding times for LMWH-DOX-Lip and LMWH-ss-DOX-Lip groups compared with the LMWH-sol group.

Fig. 12 Bleeding time evaluation via cutting tail test of LMWH-sol and LMWH modified liposomes with indicated LMWH concentration.

4. Discussion

4.1 Rational design of LMWH functionalized redox-responsive liposomes

Along with the advancements of various functional materials and new therapeutic techniques,^34–43 many meaningful efforts have been made to overcome tumor metastasis.⁴⁴ Herein, we designed and synthesized LMWH functionalized reduction responsive DOX loaded liposomes for attenuation of tumor metastasis.

LMWH is a natural sulfated glycosaminoglycan which is abundant in human tissues. Both heparin and LMWH are capable of anti-metastatic activities, and numerous useful studies have certified their positive effects.^45–47 Compared with natural heparin, LMWH has lower binding properties for circulating and cellular proteins, endowing LMWH with strong advantages of longer plasma half-life, less ability to inactivate thrombin, lower risk of bleeding and heparin-induced thrombocytopenia, etc.⁴⁸ Thus, LMWH attracts more attention in research and clinical use.

In this work, LMWH was chemically linked to the liposome surface with the synthesis scheme presented in Fig. 2. This rational design is based on the following two points. Firstly, the direct usage of free LMWH carries a typical risk of bleeding. There have been reports that the antithrombin ability of heparin is related to the carboxyl groups on the backbone; therefore, chemical reactions with the carboxyl group could decrease its antithrombin ability,⁴⁹ lowering the risk of bleeding during usage. As shown in Table S2,† the activated partial thromboplastin time (APTT) assay revealed that the synthesized LMWH-DTSP-OSu has a shorter thromboplastin time. Secondly, for drug loaded liposomes, a typical drawback is rapid clearance from the body. The chemical structure of LMWH is full of hydroxyl groups (Fig. 1A), which could form hydrogen bonds with surrounding water molecules. Therefore, the modification of LMWH on the liposome surface could act as a “shield” to protect liposomes from elimination, with a similar effect to PEG. The pharmacokinetic data (Fig. 11) illustrated that LMWH-DOX-Lip and LMWH-ss-DOX-Lip have significantly longer t_1/2 values, 0.94 h and 0.85 h, respectively, compared with the saline group (0.21 h) and the DOX-Lip group (0.83 h). Meanwhile, modification of LMWH could also promote the cellular uptake of liposomes, and this point will be discussed in detail in the next section.

However, direct conjugation between the LMWH molecules and liposomes inhibits DOX release to some extent; as shown in Fig. 3, the cumulative release of DOX after 24 h in PBS was 57% and 65% for LMWH-DOX-Lip and LMWH-ss-DOX-Lip (no DTT), respectively, while that of DOX-Lip was 81%. This may be due to the diffusion inhibition of DOX by the LMWH “shield”. Meanwhile, the anti-tumor metastasis ability of LMWH would be also weakened due to the “stable” chemical modification (Fig. 7 and 8). Therefore, the reduction responsive disulfide bond was introduced. Owing to the high level of GSH in the cytoplasm, DOX and free LMWH could be rapidly released via breakage of the disulfide bonds.

Based on the above analysis, LMWH functionalized redox-responsive DOX loaded liposomes were prepared according to the preparation scheme shown in Fig. 2. The particle sizes of the prepared liposomes were around 110 to 120 nm. TEM images were also collected, with the fluffy sphere-like morphology of LMWH-ss-DOX-Lip, as shown in Fig. 3. According to the previous reports, nanoparticles of 20–200 nm in size can be enriched in tumor tissues through EPR effects.⁵⁰ The prepared liposomes in this work fall well in this range. Afterwards, in the presence of 10 mM DTT, mimicking the reduction conditions of GSH in tumor cells, the cumulative release of DOX could reach 79% within 24 h, enabling relatively faster release of DOX in the cytoplasm.

4.2 In vitro evaluation of the anti-metastasis effects of LMWH and the cellular uptake and cytotoxicity of drug loaded liposomes

In this work, the Transwell assay was adopted to evaluate the inhibition effects of LMWH on cell invasion and migration in vitro. As shown in Fig. 7 and 8, after treatment with LMWH-sol or LMWH functionalized liposomes, the cell invasion and migration of B16F10 cells was significantly weakened compared with the saline group. Meanwhile, owning to the breakage of disulfide bonds, more free LMWH molecules were released in the tumor cytoplasm, leading to stronger inhibition efficiency, as shown by the significant difference between LMWH-ss-Lip and LMWH-Lip (p < 0.01 for cell invasion, p < 0.05 for cell migration).

One of the reasons for the positive effect of LMWH on anti-tumor metastasis may be the inhibition of Hap expression.¹¹ An inevitable process for tumor metastasis is to pass through the extracellular matrix, which mainly consists of collagens, fibronectin and heparin sulfate proteoglycans (HSPG).⁷ HSPG is composed of a core protein and is conjugated with several side chains of heparin sulfate (HS). HS plays a crucial role in the integrity and stability of the extracellular matrix.⁴⁵ However, heparanase could specifically recognize the HS side chain and degrade HS into short oligosaccharides, leading to an increased possibility of tumor metastasis.¹¹ Fortunately, LMWH could inhibit the expression of heparanase; as shown in Fig. 9, in the presence of LMWH, the expression of Hap was significantly downregulated.

In addition to the inhibition of Hap expression, modification with LMWH can increase the cellular uptake and cytotoxicity of drug loaded liposomes. Compared with the free diffusion of drug molecules into tumor cells, liposomes require uptake via endocytosis, leading to a slower cellular uptake and higher IC₅₀ values in vitro. However, both the CLSM and flow cytometry results certified that modification of LMWH on the surface of DOX-Lip could enhance the cellular uptake, represented by the stronger fluorescence of DOX (red) in the tumor cells, as shown in Fig. 5. A competitive experiment was also conducted. Following pre-treatment with 100 μg mL⁻¹ LMWH for 0.5 h for the LMWH-ss-DOX-Lip group, the phenomenon of enhanced cellular uptake disappeared, illustrating the existence of receptor-mediated intracellular delivery for LMWH functionalized liposomes. Afterwards, the enhancement of DOX delivery into the tumor cells led to stronger anti-tumor activity for LMWH-ss-DOX-Lip, with much lower IC₅₀ values compared with DOX-Lip, as presented in Fig. 6. All these results certified the superior anti-tumor and anti-metastasis abilities of LMWH-ss-DOX-Lip in vitro.

4.3 In vivo anti-melanoma metastasis assay and possible side effects evaluation

The pulmonary melanoma metastasis assay is a commonly used model to evaluate the metastatic ability of tumor cells in vivo.⁵¹ The B16F10 cell line has high metastatic ability; after intravenous injection, B16F10 cells can accumulate and implant in lung tissues. Lung weight and tumor metastasis foci were also selected as assessment criteria. As shown in Fig. 10A and C, the metastatic ability of tumor cells in the group of LMWH-ss-DOX-Lip was the lowest compared with other experimental groups within the observation time of 21 days. This may be due to the synergistic effect of DOX and LMWH, presented as the following two points. The first point is related to killing circulating tumor cells and decreasing their invasion and migration abilities. That is, after injection with LMWH-ss-DOX-Lip, some of the functionalized drug loaded liposomes underwent cellular uptake by the circulating tumor cells of B16F10 via receptor-ligand mediated endocytosis (Fig. 5), which led to enhanced apoptosis and necrosis of circulating B16F10 cells by the chemotherapeutic drug DOX. Afterwards, the existence of LMWH attenuated the abilities and possibilities of cell invasion and migration to foreign tissues (Fig. 7 and 8). The second point is related to the treatment of previously infiltrated tumor cells. Even if some B16F10 cells infiltrated and implanted in the lung tissue, forming tumor metastasis nodes, LMWH-ss-DOX-Lip could enter these tumor tissues via EPR effects and realize fast intracellular release of DOX via the breakage of disulfide bonds, achieving the growth inhibition and regression of these tumor nodes. Thus, the group of LMWH-ss-DOX-Lip has the strongest anti-metastasis and anti-tumor activities.

Mice body weights were recorded during therapy to characterize the general toxicity of chemotherapeutics. As shown in Fig. 10D, for the LMWH-ss-DOX-Lip group, the body weight of the mice remained constant, while those in the DOX-sol or DOX-Lip groups decreased significantly, indicating that LMWH-ss-DOX-Lip has a very mild general toxicity compared with DOX-sol or DOX-Lip. Meanwhile, the bleeding risk of different formulations was also evaluated; it was found that LMWH-DOX-Lip and LMWH-ss-DOX-Lip had decreased risk compared with LMWH sol alone (Fig. 12). Afterwards, routine blood examination was performed to evaluate the potential general blood toxicity of the drug loaded liposomes; as shown in Fig. S2,† white blood cell number, red blood cell number, platelet number and hemoglobin concentration had no obvious differences between the saline group and other experimental groups, indicating no obvious blood toxicity.

Overall, the delivery system of LMWH-ss-DOX-Lip could exhibit higher anti-tumor and anti-metastasis abilities and lower side effects. This positive efficiency was attributed to the rational design of drug carriers with a combination of LMWH moieties and DOX loaded liposomes via reduction responsive modification.

It is worth mentioning that incorporating other specific active ligands of B16F10 cells besides LMWH in the design of drug delivery systems, such as CD44 antibody or RGD peptide, could realize better performance for the prevention of tumor metastasis. The dual active targeting effect would enhance the recognition and cellular uptake of circulating B16F10 cells, leading to higher cytotoxicity. Meanwhile, this effect would also lead to higher drug accumulation in metastatic tumor tissues.

5. Conclusions

A novel delivery system of liposomal DOX was successfully fabricated with LMWH modification via a labile disulfide linkage. LMWH and DOX exhibited synergistic effects on the attenuation of tumor metastasis. The reduction responsive and shell-sheddable design of LMWH-ss-DOX-Lip enabled enhancement of the cellular uptake and cytotoxicity of DOX and better performance of LMWH for the prevention of invasion and migration of tumor cells. In vivo pulmonary melanoma metastasis assays confirmed the superior anti-tumor and anti-metastasis efficiencies of LMWH-ss-DOX-Lip. Possible side effects were also evaluated with body weight, bleeding time and blood routine examination as criteria, indicating that the reduction-responsive LMWH modified liposomes have mild adverse effects. Therefore, we conclude that the novel delivery system of LMWH-ss-DOX-Lip is a promising vehicle for tumor treatment and the attenuation of metastasis.

Acknowledgements

This work was financially supported by NSF of China (grant No. 81302730) and the Program of the “333 high-level personnel training project (No. BRA2015392)” Young leaders in science and technology of Jiangsu Province and the Public Technology Service Center of Nanodrug Preparation and Evaluation of Jiangsu Province. The authors express their gratitude to the support of Jiangsu Key Laboratory of Carcinogenesis and Intervention (China Pharmaceutical University) for assistance with cell and animal experiments.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra27227k

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