Targeted delivery of doxorubicin and vincristine to lymph cancer: evaluation of novel nanostructured lipid carriers in vitro and in vivo

Abstract

Purpose: Lymph cancers are heterogeneous malignancies of the hematopoietic and lymphoid tissues. Doxorubicin (DOX) and vincristine (VCR) are commonly used anti-cancer chemotherapeutic drugs, but their clinical uses are associated with dose-limiting systemic toxicity.

Methods: In the present study, DOX and VCR were encapsulated into nanostructured lipid carriers (NLCs) and used them to treat B-cell lymphoma cells through the targeted delivery of DOX and VCR to lymph cancer animal model.

Results: DOX and VCR encapsulated NLCs (DOX/VCR NLCs) demonstrated controlled drug release under physiological conditions. In addition, DOX/VCR NLCs exhibited the highest cytotoxicity and synergistic effect of two drugs in B-cell lymphoma cells and the best anti-tumor effect in vivo.

Conclusion: DOX/VCR NLCs were proved to be more efficacious than the equivalent dose of free DOX and single drug (DOX or VCR) formulation in vitro and in vivo, and significantly reduced the drug-associated systemic toxicity.

• B-cell lymphoma
• lymph cancer
• nanostructured lipid carriers
• synergistic therapy
• targeted delivery

Introduction

Approximately 70 000 people are diagnosed with B-cell lymphomas in the USA alone (Chen et al., 2010). Ninety percent are B-cell lymphomas, which cause an estimated 18 940 annual deaths (Yang et al., 2013). Diffuse large B-cell lymphoma (DLBCL) is the most frequently diagnosed lymphoid tumor, accounting for 40% of all non-Hodgkin lymphomas among adults in Western countries (Wang et al., 2015). DLBCL is also common in developing countries (Sabattini et al., 2010). Although traditional chemical therapies and bone marrow transplantations can increase survival rates and even cure certain patients, relapse and drug resistance remain a clinical challenge (Alzouebi et al., 2012). In order to enhance therapeutic efficacies and reduce adverse effects, novel therapeutics with alternative mechanisms of B-cell killing are needed (Castillo et al., 2008).

Nanomedicine is an emerging field that has shown great promise for the development of novel diagnostic, imaging and therapeutic agents for a variety of diseases, including tumor (Xiao et al., 2011). To limit some of the side effects associated with many chemotherapeutics agents such as doxorubicin (DOX) and vincristine (VCR), several nanocarrier systems have been developed (Davis & Farag, 2013; Wang et al., 2014; Cabeza et al., 2015; Li et al., 2015). These nanomaterials include lipid nanoparticles (Thakkar et al., 2015), liposomes (Hashimoto et al., 2015), polymeric nanoparticles (Prabhu et al., 2015), dendrimers (Setaro et al., 2015) and so on. It is believed that such nanocarriers will decrease the toxicity to normal organs, prolong the circulation time, slow down the metabolism of the drug and facilitate the delivery of the drug to the tumor sites via the enhanced permeability and retention effect (Keereweer et al., 2012).

It has been reported that lipid nanoparticles improve the drug absorption and bioavailability due to their diameter and absorption enhancing effect of lipids (Hejri et al., 2013). Nanostructured lipid carriers (NLCs) represent an improved generation of lipid nanoparticles (Yuan et al., 2013). They are advised to be the second lifetime of lipid nanocarriers. Contrasted to SLN and other lipid carriers, NLCs show a higher loading capability by blending a fluid lipid with the solid lipid, a higher element drug stacking can be achieved and a lower likelihood of drug expulsion during storage (Han et al., 2015).

A combination therapy with two or more chemotherapeutics for refractory cancers is a successful strategy for synergistic effects, lower toxicity and drug resistance (Zanotto-Filho et al., 2015). DOX and VCR are commonly used anti-B-cell lymphoma chemotherapeutic drugs, but their clinical uses are associated with dose-limiting systemic toxicity. In the present study, DOX and VCR were encapsulated into NLCs and used them to treat B-cell lymphoma cells through the targeted co-delivery of DOX and VCR. This system was expected to enhance synergistic effects for B-cell lymphoma, bring about a new combination therapy strategy for the lymph cancer.

Materials and methods

Materials

VCR was purchased from Shaanxi Pioneer Biotech Co., Ltd. (Xi’an, China). Doxorubicin hydrochloride (DOX·HCl) was supplied by Dalian Meilun Biology Technology Co., Ltd. (Dalian, China). COMPRITOL®888 ATO was generously provided by Gattefossé USA (Paramus, NJ). Polyoxyl castor oil (Cremophor ELP) was donated by BASF (Ludwigshafen, Germany). Didecyldimethylammonium bromide (DDAB) was purchased from Ziyi Reagent Factory (Shanghai, China). The human DLBCL cells (LY1 cells) were obtained from the American type culture collection (Manassas, VA). All the solvents used were of analytical reagent grade.

Animals

BALB/c mice (6–8 weeks old, 18–22 g weight) were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All animal experiments complied with the Animal Management Rules of the Ministry of Health of the People’s Republic of China

DOX/VCR NLCs construction

Prior to the NLCs preparation, the DOX·HCl was stirred with twice the molar amount of TEA in DMSO for 12 h to obtain lipophilic DOX base (Han et al., 2014). COMPRITOL® 888 ATO Stearic acid, Cremophor ELP, DOX base and VCR (DOX versus VCR = 1:1, w/w) were melting and dissolving at 65 °C as the lipid phase. A cold aqueous phase was prepared by dissolving DDAB in 50 mL distilled water. The lipid phase was rapidly injected into the aqueous phase. DOX/VCR NLCs were harvested by stirring in an ice bath for another 10 min. DMSO was removed by the dialyzed method. The redundant stabilizers were removed by centrifugation at 1000 g, 4 °C for 30 min. The pellet was vortexed and resuspended in Milli-Q water, washed three times, filtered through a 0.45-μm membrane. The obtained NLCs were stored at 2–8 °C.

Single DOX or VCR containing NLCs (DOX NLCs or VCR NLCs) or blank NLCs were prepared by the same method described earlier.

NLCs characterization

The particle size and zeta potential of different NLCs were measured using a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). The entrapment efficiency (EE) of DOX (EE_DOX) in NLCs was determined by high performance liquid chromatography (HPLC, Agilent 1100, Santa Clara, CA) with a Zorbax SB-C₁₈ column (150 mm× 4.6 mm, 5 μm). The mobile phase consisted in a mixture of 0.05 mol/L potassium dihydrogen phosphate solution (pH adjusted to 7.5) with acetonitrile (70:30 v/v). The flow rate was maintained at 1.0 mL/min. The effluents were monitored at λ_Ex = 480 nm and λ_Em = 560 nm.

The EE of VCR (EE_VCR) in NLCs was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). The detection was carried out at λ = 275 nm.

EE was calculated by the following equation:

NLCs release study

DOX/VCR NLCs and other kinds of NLCs were added into dialysis bags (Shao et al., 2015). Then, the dialysis bags were incubated with 20 mL release medium at 37 °C with stirring at 100 rpm. At predetermined time intervals, the NLCs suspensions were centrifuged and the amount of drug released in the supernatant was analyzed. The amount of DOX was measured by using HPLC, and the DDP content was determined by ICP-OES mentioned in the section “DOX/VCR NLCs construction”.

Cytotoxicity

The cytotoxicity of DOX/VCR NLCs with the different DOX to VCR ratios (5/1; 2/1; 1/1; 1/2; 1/5; w/w), DOX NLCs, VCR NLCs, DOX solution and VCR solution were evaluated by MTT assay (Namvar et al., 2015). The LY1 cells were grown in the RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO₂ humidified atmosphere. The confluent monolayer cells were harvested and seeded into microplates for 24 h in the incubator for cell attachment and cell recovery after trypsinization before subjected to NLCs treatment and incubation for 48 h. Then, MTT solution (5 mg/mL) was added to each well and cells were incubated for 4 h. Two hundred microliters DMSO was added to each well to dissolve the MTT formazan crystals. The optical density (OD) of formazan product was measured at 570 nm. Cells without the addition of MTT reagents were used as a blank control. Cell viability was expressed as the percentage of MTT counts of treated cells relative to those without MTT treatment. The IC₅₀ was the concentration that caused 50% inhibition of cell viability and was calculated.

In vivo anti-tumor efficacy

LY1 cells were inoculated into the right armpit of BALB/c mice. When tumor volume reached 90–110 mm³, transplanted mice were randomly divided into six groups (six per group) separately.

Mice were injected intravenously every 3 days (d) with DOX/VCR NLCs, DOX NLCs, VCR NLCs, DOX solution, VCR solution and 0.9% saline as control. Twenty-one days later, all the mice were sacrificed by cervical dislocation and the tumor tissue samples were taken out.

Tumor size was measured by a digital caliper every 3 d. Tumor volume (mm³) was calculated by V = L × W²/2, where L and W are the longest and shortest diameter of tumors.

The weight of each mouse was measured every 3 d after injection to determine the body weight lost of different groups.

Statistical analysis

Results were presented as mean ± SD. Statistical comparisons were made by Student’s t-test or ANOVA analysis. The accepted level of significance was p < 0.05.

Results and discussion

NLCs characterization

The size of DOX/VCR NLCs was 96 nm. Blank NLCs was 94 nm. These data illustrated that the loading of drugs did not increase the size significantly. It has been reported that the nanoparticles with the size less than 200 nm can significantly accumulate in tumor by “filtration” mechanism (Xu et al., 2009; Duan et al., 2015). Small particles are also minimal endocytosis by macrophages, so destruction and clearance could be minimized, and the nanocarriers were desirable for in vitro and in vivo drug delivery (Accardo et al., 2012; Wang et al., 2013). The zeta potentials of DOX/VCR NLCs were +21 mV. The cationic property the NLCs is due to the DDAB as surfactant used during preparation. Positive charge of the nanocarriers could exploit the negative charged cells due to the electrostatic interactions, thus increase the cellular uptake (Xu et al., 2012; Vighi et al., 2012). The EE of DOX and VCR loaded in DOX/VCR NLCs and single drug NLCs were above 80%. High EE could guarantee the therapeutic efficiency of the nanomedicine (Table 1).

Table 1. NLCs characterization.

Characters	DOX/VCR NLCs	DOX NLCs	VCR NLCs	Blank NLCs
Size (nm)	95.8 ± 2.1	96.2 ± 2.3	94.9 ± 2.6	94.1 ± 1.7
Potential (mV)	+21.3 ± 2.8	+16.9 ± 1.9	+27.1 ± 2.3	+20.9 ± 1.6
EEDOX (%)	87.9 ± 2.4	88.3 ± 2.7	–	–
EEVCR (%)	83.3 ± 2.1	–	85.8 ± 1.9	–

NLCs release study

DOX and VCR release profiles of DOX/VCR NLCs and single drug NLCs are shown in Figures 1 and 2. NLCs showed the sustained-release behavior of both drugs. We consider over 80% of accumulated releases as the complete release of drugs. DOX releases were completed at 16 h, while VCR release was slower, at 48 h. The sustained-release of drugs from NLCs can continuously contribute anti-tumor effect for relatively long period of time (Momoh et al., 2013; Wang & Xia, 2014). This may help with the anti-tumor progress.

Figure 1. Accumulated release of DOX from DOX/VCR NLCs, and DOX NLCs.

Figure 2. Accumulated release of VCR from DOX/VCR NLCs, and VCR NLCs.

DOX to VCR ratio determination in DOX/VCR NLCs

To determine the best DOX to VCR ratio of the DOX and VCR loaded in NLCs, the best synergy effect of these two drugs in the formulations must be achieved. The cytotoxicity was evaluated by MTT assay in LY1 cells, and the IC₅₀ values of DOX/VCR NLCs with the different DOX to VCR ratios (5/1; 2/1; 1/1; 1/2; 1/5; w/w) are summarized in Table 2.

Table 2. DOX to VCR ratio determination in DOX/VCR NLCs.

DOX to VCR ratios (w/w)	IC50 of DOX (μg/mL)	IC50 of VCR (μg/mL)
5/1	2.15	0.43
2/1	1.92	0.96
1/1	0.26	0.26
1/2	0.85	1.71
1/5	0.38	1.91

The DOX/VCR NLCs showed the lowest IC₅₀ (0.26) when DOX to VCR ratio was 1/1, showing the best synergism effect. It could be concluded that the synergistic effect of DOX and VCR contained in NLCs at the weight ratio of 1/1 was the best formulation in the present study.

Cytotoxicity assays

In vitro cytotoxicity of DOX/VCR NLCs, DOX NLCs, VCR NLCs, DOX solution and VCR solution was evaluated by MTT assay in LY1 cells. Table 3 shows the IC₅₀ values of drugs solutions and NLCs formulations in LY1 lymph cancer cells. The IC₅₀ values of DOX and VCR solution were 7.51 and 9.82 μg/mL, respectively. The IC₅₀ values of DOX and VCR NLCs were 2.63 and 3.31, respectively. These results were threefold dose advanced than the solutions. This can contribute to the advantage of NLCs that improved the drug bioavailability. DOX/VCR NLCs has the IC₅₀ of 0.26/0.26 of the two drugs. More than 10-fold of IC₅₀ values of DOX/VCR NLCs over DOX NLCs and VCR NLCs in reducing viability of lymph cancer cells was observed, accounting for the highest anti-tumor activity and exhibited the significantly synergy effect.

Table 3. DOX to VCR ratio determination in DOX/VCR NLCs.

Formulations	IC50 of DOX (μg/mL)	IC50 of VCR (μg/mL)
DOX solution	7.51	–
VCR solution	–	9.82
DOX NLCs	2.63	–
VCR NLCs	–	3.31
DOX/VCR NLCs	0.26	0.26

In vivo anti-tumor efficacy

According to the results of in vitro cytotoxicity assays, the in vivo anti-tumor studies of different formulations were applied in mice bearing LY1 cells lymph cancer model. Figure 3 shows the tumor growth curves. The results could be summarized as follows: On the one hand, NLCs formulations showed better efficacy than free drug solutions (p < 0.05); on the other hand, tumor growth was more significantly inhibited by DOX/VCR NLCs than other NLCs groups (p < 0.05). The most obviously tumor regressions were observed in the double drug containing NLCs group, showing the synergistic effect and the excellent delivery ability of the novel NLCs.

Figure 3. Tumor volume changes after treatment with different formulations in mice bearing lymph cancer xenograft.

Systemic toxicity during the cancer treatment is a severe side effect. Changes in the body weight of tumor-bearing mice after injections can indicate the systemic toxicity. The body weights of DOX/VCR NLCs, DOX NLCs and VCR NLCs groups slightly increased, which might due to the low toxicity of NLCs and slight increase of the tumor weight (Figure 4). Noticeable body weight losses were observed in DOX solution, VCR solution and saline control groups. Also, reduction in food intake, energy sag and inactive in moving were observed in these three groups during the test. The results demonstrated the reduced systemic toxicity in the NLCs formulations.

Figure 4. Body weight changes after treatment with different formulations in mice bearing lymph cancer xenograft.

Conclusions

DOX/VCR NLCs exhibited the highest anti-tumor activity and the significantly synergy anti-lymph cancer effect in vitro and in vivo. This system could achieve targeted co-delivery of DOX and VCR, overcome the drug resistance of the single drug preparation and reduce the systemic toxicity. Also, NLCs formulation could target the lymph cancer site due to its small particle size and positive surface charge. The resulting nanomedicine could function as a promising targeted delivery system for the treatment of lymph cancers.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.