Introduction

Melanoma is a common type of skin tumour. The limited effectiveness of radiotherapy and chemotherapy as treatment options [1] render melanoma difficult to cure. Currently, the use of RNA interference (RNAi) to inhibit melanoma development represents a novel treatment strategy. Small interfering RNAs (siRNAs), first discovered by the British scientist David Baulcombe and his team in 1999 [2], are double-stranded RNA molecules that play important roles in the process of RNA interference. However, naked siRNA has a short biological half-life, is readily degraded by enzymes, shows low bioavailability, and lacks target specificity. Moreover, given their hydrophilic nature and negative charge, siRNA molecules are not readily transported across cell membranes, and hence carrier-mediated transfer is often required for the effective transformation of cells. Vectors used to mediate such trans-membrane transfer can be divided into viral and non-viral types. Of these, viral vectors are characterised by a high transduction and expression efficiency, but they tend to have a limited gene-loading capacity. Moreover, these vectors can cause inflammation and immune reactions in the body, thereby limiting their utility [3]. Common viral vectors used for gene therapy are adenoviruses and retroviruses [4]. In contrast, non-viral vectors have the characteristics of lower immunogenicity and cytotoxicity, greater transport capacity, and ease of preparation; thus, they are considered promising as vectors [5]. Among these, cationic liposomes are the most commonly used carrier materials.

The complexes generated through the direct binding of cationic liposomes and siRNA are inherently unstable, and consequently, their transmission efficiency is low. To overcome this challenge, studies have focussed on developing modified liposomes characterised by greater stability, among which are liposome–protamine–DNA (LPD–NP) composite nanoparticles. Studies have shown that LPD–NP nanoparticles can effectively deliver siRNA into recipient cells. Furthermore, Li et al. [6, 7] has successfully demonstrated the anti-tumour effect of these nanoparticles. Protamine, a polycationic peptide, which has anti-tumour, anti-viral, and anti-bacterial effects [8], is the only registered heparin antidote that is resistant to the anti-coagulant effect of heparin or synthetic anti-coagulants. It has been certified by the US Food and Drug Administration (FDA) for clinical use. However, protamine at high concentrations can alter the electrical properties of protamine–DNA complexes, thereby affecting their combined effect with cationic liposomes. In addition, the CpG motifs in plasmid DNA render the nanoparticles highly immunogenic [9]. To reduce the immunotoxicity of nanoparticles and improve the preparation of nanoparticles, hyaluronic acid (HA) is added. HA is present in the extracellular matrix of most connective tissues. It is inherently electronegative, has low toxicity, and is devoid of immunostimulatory CpG motifs [10]. Therefore, it has been examined for use as a carrier for administration via different routes, including the ocular, nasal, and oral routes [11]. Furthermore, studies have indicated that low-molecular weight HA has an inhibitory effect on tumour metastasis [12]. Accordingly, to enhance the preparation of nanoparticles, we used HA to replace plasmid DNA.

Currently, studies on apoptosis have focussed on the development of tumours. It is believed that cancer is caused by abnormal processes in the cell cycle and apoptosis [13]. Furthermore, it has been demonstrated that one of the characteristics of cancer cells is their ability to evade apoptosis and undergo repeated replication [14]. In this regard, survivin, a unique member of the IAP family of apoptosis inhibitors, has been found to play multiple roles in regulating cell division and apoptosis, maintaining cell proliferation and survival under adverse conditions, and inducing resistance to different anti-cancer treatments [15]. Studies using tumour cell models have revealed that over-expression of survivin is associated with cell protection and apoptosis inhibition; moreover, it induces resistance to the effects of different pro-apoptotic proteins and chemotherapeutic drugs [16]. Moreover, Lu et al. [17] reported that the expression of survivin was strongest in melanoma cells. They also demonstrated that during the tumour proliferation phase, that is, during tumour angiogenesis, survivin is significantly expressed in vascular endothelial cells, indicating that it plays a direct role in tumour angiogenesis and canceration [18]. Furthermore, Ding et al. [19] found no significant difference in the distribution of cytoplasmic survivin in moles and melanomas. Whereas, although nuclear-localised survivin is expressed in melanomas, it is not similarly expressed in moles, and the activity of cytoplasmic survivin appears to be unrelated to survival and relapse rates. More importantly, survivin is highly expressed in different types of cancer tissues; it can also be detected during the developmental of embryonic organs, but not in normal tissues [13]. Therefore, survivin appears to be of considerable significance as a target for gene silencing.

In summary, when cationic liposomes are combined with siRNA alone, polymerisation is highly unstable, whereas when LPD nanoparticles are used as a carrier, plasmid DNA may promote excessive immunogenicity. Therefore, in this study, we used HA instead of plasmid DNA to construct a novel type of cationic liposome carrier that can carry siRNA targeting melanoma survivin and evaluated the efficacy of this carrier and the potential of this target.

Materials and methods

Materials

N-[1-(2,3-Dioleoyloxy) propyl]-N,N,N-trimethylammonium methyl-sulphate (DOTAP) was purchased from Shanghai Sixin Biotechnology Co. Ltd. Protamine, HA, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were purchased from Shanghai Yuanye Biotechnology Co. Ltd. Dulbecco’s modified Eagle medium (DMEM) and foetal bovine serum (FBS) were purchased from Sai Ai Mei Cell Technology Co., Ltd. Diethyl pyrocarbonate (DEPC) was purchased from BeiJing BoAo TuoDa Technology Co., Ltd. Double antibody was purchased from Gibco (USA). Anti-survivin antibody and goat anti-rabbit IgG (H + L) HRP conjugate were purchased from Abcam (USA). Glycine, sodium dodecyl sulphate (SDS), Tris-base, acrylamide, ammonium persulphate and tetramethylethylenediamine (TEMED) were purchased from Amresco (USA). The siRNA targeting survivin gene (5′-CCG AGA ACG AGC CUG AUU UTT-3′) and negative control siRNA (5′-UUC UCC GAA CGU GUC ACG UTT-3′) were synthesised by Suzhou Jima Gene Co., Ltd.

Cell

B16F10 mouse melanoma cells [20] were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. All other reagents used in this study were of analytical grade.

Animals

Per Wang et al. [21], 6–8-week-old female C57BL/6 mice were used in the animal experiments. The mice were purchased from the Experimental Animal Centre of Heilongjiang University of Traditional Chinese Medicine. The animal experiment protocol conformed to the Guiding Principles for the Care and Use of Experimental Animals.

Instrument

Rotary evaporator OSB-2200 was purchased from Japan EYELA Co., Ltd. Ultrasonic cell disruption instrument was purchased from SONICS USA. Enzyme-linked immunosorbent assay kit SYNERGY H1 was purchased from American Boteng Instrument Co., Ltd. The potential particle size analyser Nano-ZS90 was purchased from Malvern (UK). Transmission electron microscope JEM-2100 was purchased from JEOL Ltd. Fluorescent inverted microscope was purchased from Leica (Germany). Fluorescence quantitative PCR instrument ABI7500 was purchased from Applied Biosystems (USA).

Methods

Preparation of siRNA cationic liposome nanoparticles

To prepare cationic liposomes, DOTAP:cholesterol at a ratio of 1:1 was dissolved in chloroform, and a rotary evaporator was used to remove the organic solvent until a thin lipid film was formed on the bottle wall. The mixture was then treated with a sufficient amount of DEPC water to wash the film. Following incubation at room temperature (25 °C) for 1 h, liposome solution was prepared at a concentration of 40 mM. Following ultrasonication, the solution was passed through a 0.22-μm microporous membrane filter. The filtration step was repeated 5–10 times to obtain cationic liposomes.

We then prepared the HA-siRNA-protamine complex. siRNA and HA were mixed at a ratio of 1:1, shaken thoroughly, and an appropriate amount of protamine was added, followed by incubation at room temperature (25 °C) for 10 min to obtain the HA–siRNA–protamine complex. The cationic liposomes prepared earlier were added to the HA–siRNA–protamine complex, and then an appropriate amount of DSPE-PEG2000 was added after 10 min. The mixture was then placed in a water bath at 50 °C for 10 min to obtain siRNA cationic liposome nanoparticles.

Optimisation of cationic liposomes

For different mass ratios of HA–siRNA and protamine of 0.8, 0.85, 0.9, 0.95, 1.0, and 1.05, HA–siRNA and protamine were weighed accordingly, mixed, and incubated at room temperature for 10 min. Particle size and zeta potential of each complex were measured using a potentiometric particle size analyser.

The cationic liposomes of different concentrations (10, 20, 30, 40, 50, 60, and 70 μL) were mixed with the HA–siRNA–protamine complex containing 25 μg of survivin siRNA; the mixture was left at room temperature for 10 min. Similarly, the particle size and zeta potential of the obtained liposome nanoparticles were measured using a potentiometric particle size analyser. Thus, the optimal concentration of the cationic liposomes and the ratio of HA–siRNA-to-protamine were determined. The siRNA liposome nanoparticles were prepared according to the optimised protocol.

Physico-chemical characterisation of cationic liposomes

Zeta potential and particle size of the nanoparticles were determined using the Nano-ZS90 particle size analyser (Malvern, UK) at room temperature.

Five microlitres of siRNA cationic liposome nanoparticles was applied on to a copper mesh and dried at room temperature. The copper mesh was observed under a JEM-2100 transmission electron microscope (JEOL Ltd., Tokyo, Japan) to determine the form of the liposomes.

Determination of the loading efficiency of liposomes

UV spectroscopy

Fluorescently labelled siRNA (FAM–siRNA) was dissolved in DEPC to prepare the FAM–siRNA solution. An appropriate amount of the FAM–siRNA solution was diluted with DEPC-treated water to obtain 0.05, 0.10, 0.15, 0.20, 0.25, and 0.30 μg/μL standard solutions with different concentration gradients. The absorbance of the solution was measured using a microplate reader at 492 nm. A standard curve of absorbance (Y-axis) versus FAM–survivin siRNA concentration (X-axis) was generated.

An appropriate amount of the FAM–siRNA solution to be tested was centrifuged in a centrifuge tube and the absorbance of the supernatant was measured at 492 nm using a microplate reader. Free FAM–siRNA concentration was calculated using the standard curve. Encapsulation efficiency was calculated by subtracting the amount of FAM–siRNA present in the supernatant from the total input FAM–siRNA.

Stability test

To detect the encapsulation effect of the nanoparticles, the cationic liposome nanoparticles prepared according to the optimised protocol and naked survivin siRNA, as a control, were subjected to agarose gel electrophoresis. In addition, the siRNA cationic liposome nanoparticles were sealed and stored in a refrigerator at 4 °C for 6 months to verify the encapsulation efficiency of the synthesised nanoparticles.

In vitro assays of the siRNA liposome nanoparticles

Cell viability assay

B16F10 melanoma cells were grown in DMEM containing 10% phosphate-buffered saline (PBS) and 1% double antibody at 37 °C with 5% CO2 in a humidified incubator. After incubating the cells in a 96-well plate for 24 h, the complete medium was discarded, and the cells were incubated in fresh DMEM without FBS for another 24 h. The siRNA cationic liposome nanoparticles were added to the 96-well plate at gradient concentrations of 20, 40, 60, 80, and 100 nM after 24 h. The cell culture with no siRNA was used as the negative control. All treatments were performed in triplicate. Following incubation, 20 μL of MTT solution was added to each well in dark. Following 4 h of incubation, the supernatant was discarded and 150 μL of dimethyl sulfoxide (DMSO) solution was added to each well and shaken for 10 min to dissolve the crystals. The optical density (OD) of the solution in each well was measured at 490 nm by enzyme-linked immunosorbent assay using the Synergy™ H1 multi-mode microplate reader (American Boteng Instrument Co., Ltd., Winooski, VT, USA). Inhibition rate (IR %) of tumour cell proliferation was calculated using the following formula:

$$ IR\left(\%\right)=\left(1-\frac{\mathrm{the}\ OD\kern0.6em value\kern0.5em of\kern0.5em do\sin g\kern0.5em group}{the\kern0.5em OD\kern0.5em \mathrm{value}\kern0.5em of\kern0.5em control\kern0.5em group}\right)\times 100\% $$

Cell scratch assay

B16F10 melanoma cells in the logarithmic growth phase were digested to prepare a cell suspension. The cells were seeded at a density of 4 × 105 cells per well in a six-well cell culture plate. After culturing overnight, the tip of a pipette was used to scratch the bottom of the wells from one end to the other. The medium was discarded, and free cells were washed and removed using sterile PBS. The cells were then cultured in DMEM containing 1% FBS with or without siRNA liposome nanoparticles. The cell culture without nanoparticles was used as the negative control. The results of cell scratch assay were observed 24 h post incubation.

Cellular uptake assay

B16F10 melanoma cells were used to prepare cell suspensions. The cells were then seeded in a six-well plate at a density of 4 × 10 5 cells per well. The cells were then cultured in DMEM containing 1% FBS with or without FAM–survivin siRNA liposome nanoparticles. The culture without nanoparticles was used as the negative control. The cellular uptake of nanoparticles after 12 and 24 h was observed under a fluorescent inverted microscope (Leica).

In vivo study of the siRNA liposome nanoparticles

Establishment of a melanoma mouse model

B16F10 melanoma cells were used to prepare cell suspensions, which were subcutaneously injected into mice. The mice were randomly divided into the following four groups: subcutaneous injection of normal saline (negative control), siRNA liposome (siRNA liposome), siRNA only (naked siRNA), and liposome only (NC siRNA liposome). After observing changes in the modelling site of mice, drugs were subcutaneously administered based on the group every 2 days. When the tumour grew to 20 mm in size, the mice were sacrificed by neck dislocation and the subcutaneous tumour tissue was separated by surgery.

Haematoxylin-eosin (HE) staining

The surgically isolated subcutaneous melanoma tissue was placed in 4% neutral formaldehyde fixative and stored at room temperature for 24 h. The tissue blocks were dehydrated, embedded in paraffin, sectioned, and stained with H&E. A drop of neutral gum was applied to the stained melanoma tissue sections, covered with a cover slip, and observed using an optical microscope.

Reverse transcription–polymerase chain reaction (RT-PCR)

The mRNA expression of the survivin and β-actin genes was detected by RT-PCR. The β-actin gene was used as the internal control to normalise the expression of survivin gene. Primer sequences used for RNA amplification were as follows: β-actin, forward: 5′-CCGTAAAGACCTCTATGCCAACA-3′; reverse 5′-GGGGCCGGACTCATCGTA-3′; survivin, forward: 5′-CGGAGGTTGTGGTGACGC-3′; reverse: 5′-GGTAGGGCAGTGGATGAAGC-3′.

Total RNA was extracted using TRIzol, and the resulting RNA precipitate was dissolved in DEPC-treated water. The concentration and purity of the RNA were determined by spectrophotometry. The total RNA was mixed with primers, 10× PCR buffer, dNTPs, and RNase inhibitor, and then incubated in a water bath at 25 °C for 10 min. Following this, reverse transcriptase enzyme was added to the mixture to synthesise cNDA, using the amplified RNA. Quantification was performed using the ABI7500 fluorescence quantitative PCR instrument (Applied Biosystems, USA). The data were analysed using the 2-△△ CT method to quantify gene expression.

Western blotting

Bovine serum albumin (BSA) solution was diluted with PBS to gradient concentrations of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μg/mL, and then added to a 96-well plate. Each gradient concentration was set in triplicate. Coomassie Brilliant Blue solution was added to the wells and mixed, and the absorbance of the solution was measured at 595 nm on a microplate reader. A standard curve was prepared with the BSA concentrations as the abscissa and the OD values as the ordinate, and the regression equation obtained was as follows: Y = 0.01772X + 0.011. A regression ratio (R2) of 0.9968 indicated that the linear relationship between the BSA concentration and OD value was good and in the concentration range of 0–20 μg/μL.

The melanoma tissue was added to cell lysate buffer, centrifuged for 5 min at 4 °C, and then the supernatant was collected. The absorbance of the supernatant was measured using a microplate reader to determine the protein content in the sample. The concentration of proteins in each treatment group was adjusted using the lysate buffer.

The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (PVDF membrane); then, the membranes were blocked with 5% skim milk on a decolourising shaker for 1–2 h. The membranes were then incubated with the primary antibodies (1:100) overnight and secondary antibodies (1:10,000) for 1 h. The grey scale patterns of the protein bands were analysed using an imaging system, and the ratio of grey value of survivin to the internal reference was used to estimate the relative expression of survivin.

Statistical analysis

Data are presented as mean ± SD, and the statistical significance was determined using the analysis of variance. Results with a P value of <0.05 were considered significant.

Results

Optimisation of cationic liposomes

The particle size and zeta potential of HA–siRNA and protamine complexes with different mass ratios were determined, and the results are shown in Table 1 and Fig. 1a. When the HA–siRNA-to-protamine mass ratio was 0.9, the composite had the largest particle size and was generally neutral. As the specific gravity of HA–siRNA increases, the particle size and zeta potential of the complex decreased rapidly. Hence, we considered 1.0 as the optimal mass ratio of HA–siRNA and protamine, and with this ratio, the complex was a small negatively charged particle.

Table 1 Determination of particle size and zeta potential of small interfering RNA (siRNA)–liposomes with varying mass ratios of hyaluronic acid (HA)–siRNA to protamine. Each value represents mean ± SD
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Fig. 1
figure 1

Formulation optimisation of small interfering RNA (siRNA) cationic liposome nanoparticles. a Changes in the complex of HA–siRNA and protamine with different mass ratios. HA–siRNA and protamine were mixed according to the ratio, and the particle size and zeta potential of each complex were measured leaving the sample at room temperature (25 °C) for 10 min. b The effect of different qualities of cationic liposomes on the complex. Liposomes of different qualities were added to the complex, and particle size and zeta potential of the complex were measured after leaving the sample at room temperature for 10 min. c The morphology of nanoparticles was observed under a transmission electron microscope. d Standard curve of absorbance (Y) versus FAM–survivin siRNA concentration (X). e Agarose gel electrophoresis was performed with the nanoparticles and naked siRNA. The results showed that the encapsulation effect of survivin siRNA cationic liposome nanoparticles was good

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Next, cationic liposomes of different masses were added to the quantitative HA–siRNA–protamine complex, and the particle size and zeta potential of the obtained liposome nanoparticles were measured. The results are shown in Table 2 and Fig. 1b. With an increase in the liposome quantity, the overall particle size increased initially and then decreased, whereas the zeta potential continued to increase. We chose 50 μL as the optimal amount of cationic liposomes for nanoparticle synthesis; the size of the nanoparticles synthesised was 127.1 nm and zeta potential was 43 mV.

Table 2 Determination of particle size and zeta potential of hyaluronic acid (HA)– small interfering RNA (siRNA)–protamine complex with varying amounts of liposomes. Each value represents mean ± SD
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Physico–chemical characteristics of the nanoparticles

According to the optimised amount of each substance, siRNA cationic liposome nanoparticles were prepared, and the particle size of the nanoparticles was 131 and zeta potential was 45 mV.

The prepared siRNA cationic liposome nanoparticles were irregular circular in shape, as shown in Fig. 1c.

Encapsulation efficiency of cationic liposomes

The absorbance of the FAM–siRNA solution of gradient concentrations was recorded; the standard curve formula was Y = 1004X + 177.1 (R2 = 0.9973). The results are shown in Table 3 and Fig. 1d; the absorbance showed a linear relationship with the FAM–survivin siRNA concentration in the range of 0.05–0.3 μg/μL. Precision and reproducibility of experiments were measured; the RSD was 1.25% and 1.93%, indicating that the precision and reproducibility were good. The absorbance of the solution was substituted into the standard curve formula to obtain the free FAM–survivin concentration. Subsequently, the encapsulation rate of FAM–survivin siRNA-containing cationic liposome nanoparticles was 85.07%, proving that liposomes have a good encapsulation rate for siRNA.

Table 3 Absorbance of standard solutions at different concentrations
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The nanoparticles and naked siRNA were subjected to agarose gel electrophoresis. The results, shown in Fig. 1e, indicated that the nanoparticles have a good siRNA encapsulation efficiency. The siRNA cationic liposome nanoparticles were sealed and stored in a refrigerator at 4 °C for 6 months. The nanoparticles showed good stability and no leakage. The results showed that the liposomes had good encapsulation efficiency.

Viability of melanoma cells treated with siRNA nanoparticles

As shown in Table 4 and Fig. 2a, the cationic liposomes carrying siRNA inhibited the proliferation of B16F10 cells in dose- and time-dependent manners. The curve fitting of inhibition rate is shown in Fig. 2b. The half maximal inhibitory concentration (IC50) at 24 and 48 h was 109.1 and 54.2 nM, respectively. Therefore, 100 nM survivin siRNA was selected for the subsequent experiments.

Table 4 Inhibition of B16F10 melanoma cells by small interfering RNA (siRNA)–cationic liposome nanoparticles (x ± s, n = 3). Each value represents mean ± SD
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Fig. 2
figure 2

In vitro study of cationic liposome nanoparticles acting on melanoma. a Effect of small interfering RNA (siRNA) –liposome on the growth of B16F10 melanoma cell. The results showed that the proliferation of melanoma B16F10 cells treated with survivin siRNA cationic liposomes was inhibited in vitro. b Curve fitting of the inhibition rate. c Cell scratch assay to determine the effect of siRNA on cell migration using B16F10 melanoma cells treated with siRNA liposome or negative control. The result showed that the survivin siRNA cationic liposome nanoparticles have a more obvious ability to inhibit cell migration. d Uptake of fluorescein-siRNA cationic liposome nanoparticles by B16F10 melanoma cells. The results showed that B16F10 cells treated with survivin siRNA cationic liposome nanoparticles had a significant uptake effect

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Inhibition of cell migration by siRNA nanoparticles

As shown in Fig. 2c, B16F10 cells were divided into a treatment group and control group, and the scratch assay was then performed. After 24 h, the scratches in the control group disappeared at 0 and 24 h, and the cells had covered the scratch gaps. At 24 h, the treatment group remained with obvious scratches, indicating that survivin siRNA-loaded cationic liposome nanoparticles have a more obvious ability to inhibit cell migration.

Internalisation of siRNA nanoparticles by melanoma cells

The treatment of melanoma cells with fluorescent-tagged siRNA liposome nanoparticles showed strong green fluorescence inside the cells 12 h post incubation, which further increased after 24 h. However, no fluorescence was observed in the cells treated with the negative control. The results of the cell uptake experiments are shown in Fig. 2d.

Histopathology of mouse melanoma tumours

Mice were sacrificed by neck dislocation and the subcutaneous tumour tissue was isolated, and the histopathological results are shown in Fig. 3a. The weight of the isolated tissues is shown in Table 5 and Fig. 3b. The results showed that the cationic liposome nanoparticles carrying survivin siRNA can significantly inhibit the proliferation of B16F10 cells in mice. However, when survivin siRNA was administered alone, it had a slight inhibitory effect on the proliferation of B16F10 cells in mice, because survivin siRNA lacks a carrier. The tumour tissues were then H&E stained and observed. The results of H&E staining of melanoma mouse tissue are shown in Fig. 3c. The subcutaneous cells showed the typical characteristics of melanoma tumour cells, including enlarged nucleus, increased nucleoplasm proportion, and enhanced cell division. The transformed cells were in clusters and were loosely arranged, similar to a chrysanthemum cluster.

Fig. 3
figure 3

In vivo study of cationic liposome nanoparticles acting on melanoma. a The mice were sacrificed by neck dislocation and the subcutaneous tumour tissue was isolated. (1) Negative control. (2) Survivin small interfering RNA (siRNA) liposome. (3) Naked siRNA. (4) NC siRNA liposome. b Weight of the separated tissue. c Haematoxylin and eosin staining of melanoma tissue from mouse. (1) Negative control. (2) siRNA liposome. (3) Naked siRNA. (4) NC siRNA liposome. d Detection of survivin gene expression upon treatment with siRNA–liposomes using RT-PCR. The results showed that survivin siRNA-containing cationic liposomes can significantly down-regulate survivin expression compared with naked survivin. e Detection of survivin expression by western blotting. f Inhibition of survivin expression determined by western blotting. The results showed that survivin siRNA cationic liposome nanoparticles could inhibit the expression of survivin in melanoma B16F10 cells

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Table 5 Weight of separated tissues. Each value represents mean ± SD
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Inhibition of tumour growth by siRNA nanoparticles

The results showed that the siRNA-loaded cationic liposomes could significantly inhibit the proliferation of melanoma in mice. When siRNA was administered alone, the inhibition of tumour growth was not significant, probably due to the absence of a vector. When cationic liposomes were loaded with NC siRNA, a certain inhibitory effect on tumour growth was observed, and they were found to be more effective than the naked siRNA. However, when mice were administered vesicles loaded with siRNA, significant tumour growth inhibition was observed, indicating the ability of siRNA nanoparticles to inhibit melanoma.

Suppression of survivin gene expression by siRNA nanoparticles

The expression of the survivin gene upon siRNA liposome treatment was estimated by RT-PCR. The β-actin gene was used as the internal reference, and the relative expression of the survivin gene was calculated using this as the control, as shown in Table 6 and Fig. 3d. The expression of the survivin gene was down-regulated in melanoma tumour tissue obtained from the mice treated with siRNA cationic liposome nanoparticles compared with that of the negative control (expression value set to 1). The rate of inhibition of survivin expression in the siRNA treatment group was 82%, whereas that in the naked siRNA and NC siRNA groups were 28% and 5%, respectively. The results showed that the siRNA cationic liposome treatment significantly down-regulated the expression of survivin in tumour tissues.

Table 6 Detection of survivin expression in small interfering RNA (siRNA)-treated and control B16F10 melanoma cells using RT-PCR. Each value represents mean ± SD
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Suppression of survivin protein expression by siRNA nanoparticles

The expression level of the survivin protein in each group was investigated by western blotting, and the results are shown in Fig. 3e and f. The expression of the survivin protein was significantly decreased in the melanoma tumour tissue of mice treated with siRNA cationic liposome nanoparticles compared with that of the control group mice. Interestingly, the survivin protein expression in the melanoma tissue of the group treated with naked siRNA was found to be lower than that of the group treated with NC siRNA. These findings highlight the role of siRNA nanoparticles in inhibiting survivin protein expression in melanoma tumour cells.

Discussion

In this study, we had two primary objectives, namely, to prepare survivin siRNA-loaded cationic liposome nanoparticles using HA and protamine, and to examine the feasibility of using survivin as a therapeutic target in the treatment of melanomas.

A range of unique cationic liposome structures have been developed as potential drug carriers and for application in gene therapy. However, nanoparticles developed using direct combination of cationic liposomes and siRNA have been found to be unstable. Owing to their anti-tumour properties, HA and protamine are used in the preparation of cationic liposome nanoparticles to enhance their therapeutic efficacy. HA and siRNA are linked via chemical bonds to form HA–siRNA conjugates [22]. The negatively charged conjugate and positively charged protamine self-assemble and link via charges. By adjusting the ratio of protamine and conjugate, negatively charged complexes can be produced, and these self-assemble with the positively charged blank cationic lipids in response to the action of electric charges to form cationic liposome nanoparticles. The zeta potential and particle size of liposomes are important properties that can influence the overall properties of liposomes. Desai et al. [23], who examined the effect of varying particle sizes on cellular uptake using a rat in situ intestinal ring model, found that nanoparticles of diameter 100 nm had a particularly high rate of cellular uptake, whereas larger nanoparticles showed relatively poor cellular uptake. If the zeta potential of cationic liposomes is too large, this may also contribute to an increase in toxicity. Optimal siRNA cationic liposome nanoparticles have a slightly higher particle size and zeta potential, which may be achieved via the addition of DSPE-PEG2000—appropriate amounts of which contributes to an increase in the retention time of liposomes in the blood and a prolonged circulatory effect.

Survivin, the smallest member of the IAP protein family, is expressed in different malignant tumours, and its expression level is associated with disease aggressiveness and clinical outcome [24]. Indeed, survivin can have effects across multiple cellular networks, and thus targeting survivin may affect the multiple signal networks in tumour cells. Accordingly, suppressing the activity of survivin may be more advantageous than targeting individual cancer pathways [25, 26]. Moreover, survivin is also found in extracellular vesicles, which play a protective role in the adjacent cancer cells and regulate the tumour microenvironment [27]. Thus, survivin might be a prime therapeutic target in the treatment of melanoma. To date, however, there have been only a few studies that have examined the pharmacodynamics of survivin and melanoma, either in vivo or in vitro.

In this study, we selected survivin as a potential therapeutic target and studied the effects of attacking survivin in melanomas using cationic liposome nanoparticles. We found that nanoparticles targeting survivin, in vitro, can inhibit B16F10 cells proliferation in time and dose matters. Moreover, these nanoparticles inhibited the metastatic ability of melanoma cells. In the in vivo experiments, in which we injected cationic liposome nanoparticles into a mouse tumour model, the nanoparticles inhibited tumour growth and significantly reduced the expression of survivin mRNA and protein. These results indicate that cationic liposome nanoparticles can effectively inhibit melanoma cells by inhibiting survivin expression; this not only demonstrates the efficacy of cationic liposome nanoparticles in targeting survivin but also emphasises the importance of survivin as a novel target in the treatment of melanomas. We also demonstrated that inhibiting the expression of survivin is a potential mechanism of the anti-tumour effect of survivin siRNA cationic liposomes, although whether it affects other factors that inhibit cell proliferation or promotes apoptosis warrants further investigation.

In our study, the control NC siRNA-loaded liposome group also showed anti-tumour activities, although administering these liposomes had no significant effect on the expression of survivin. In this regard, we speculate that HA and protamine enter cells under the guidance of liposomes, thereby inducing an anti-tumour effect. This is consistent with the findings of previous studies [8, 12], which showed that HA and protamine have certain anti-tumour effects, and thus have potential therapeutic use. Considering these findings, it will be of interest to investigate whether the incorporation of other anti-tumour drugs or traditional Chinese medicinal constituents in siRNA-loaded cationic liposome nanoparticles would have additive co-administrative effects.

In conclusion, siRNA cationic liposome nanoparticles are highly stable and have notable properties of low immunogenicity and toxicity. Accordingly, we believe that these carriers have considerable potential as novel drug delivery systems with broad therapeutic applications.