Stimulus‐responsive nano lipidosome for enhancing the anti‐tumour effect of a novel peptide Dermaseptin‐PP

Abstract

Objective

Dermaseptin‐PP is a newly discovered anticancer peptide with a unique antitumour mechanism and remarkable effect. However, this α‐helix anticancer peptide risks haemolysis when used at high doses, which limits its further application. This study aims to prepare a pH‐responsive liposome, Der‐loaded‐pHSL, using nanotechnology to avoid the haemolysis risk of Dermaseptin‐PP and increase its accumulation in tumour sites to enhance efficacy and reduce toxicity.

Methods

The characterisation of Der‐loaded‐pHSL was carried out employing preparation. The effect of haemolysis and tumour inhibition were investigated by in vitro haemolysis assay and cytotoxicity assay. The cell uptake under different pH conditions was investigated by flow cytometry, and the effect of pH on tumour cell selectivity was evaluated. In order to evaluate the in vivo targeting and antitumour effect of Der‐loaded‐pHSL, the in vivo distribution experiment and the pharmacodynamic experiment were performed using the nude mouse tumour model.

Results

The preparation method of the Der‐loaded‐pHSL is simple, and the liposome has good nanoparticle characteristics. When Dermaseptin‐PP was prepared as liposome, haemolysis was significantly decreased, and tumour cell inhibition was significantly enhanced. Compared with ordinary liposomes, this change was more significant in Der‐loaded‐pHSL. The uptake of pH‐sensitive liposomes was higher in the simulated acidic tumour microenvironment, and the uptake showed a specific acid dependence. In vivo experiments showed that Der‐loaded‐pHSL had a significant tumour‐targeting effect and could significantly enhance the antitumour effect of Dermaseptin‐PP.

Conclusion

Der‐loaded‐pHSL designed in this study is a liposome with a quick, simple, effective preparation method, which can significantly reduce the haemolytic toxicity of Dermaseptin‐PP and enhance its antitumour effect by increasing the tumour accumulation and cell intake. It provides a new idea for applying Dermaseptin‐PP and other anticancer peptides with α‐helical structure.

The Dermaseptin family AMP Dermaseptin‐PP could significantly inhibit the growth of A549 tumour cells; Dermaseptin‐PP liposome preparation can significantly reduce the dissolution of erythrocyte; pH‐responsive liposome nanodelivery system can promote Dermaseptin‐PP accumulation in tumour sites and improve anti‐tumour efficacy.

1. INTRODUCTION

Cancer is still the primary disease threatening human health, and the antitumour situation is grim. According to the report released by the World Health Organization, there were 19.29 million new cancer cases and 9.91 million deaths worldwide in 2020 [1]. Currently, chemotherapy is still the primary means of tumour treatment. However, some traditional drugs commonly used in clinical practice still have some difficult problems to overcome, such as strong toxicity to normal cells, treatment resistance due to tumour microevolution etc [2, 3]. Therefore, there is an urgent need for more effective new drugs.

Antimicrobial peptides (AMPs) are a class of small molecular peptides with antibacterial, antifungal, anti‐tumour, and immunomodulatory activities [2, 4]. Some antimicrobial peptides with α helix structure often show excellent anticancer activity, so they are also called Anticancer peptides (ACPs) [5, 6]. Dermaseptins (DRSs) are a family of peptides that are part of the skin secretions of several Hylid frogs, particularly from the Agalychnis and Phyllomedusa families [7]. DRSs are classified as Antimicrobial peptides (AMPs) because they show efficacy in vitro against some Gram‐positive and Gram‐negative bacteria, parasites, yeast, protozoa, and viruses and show immunomodulation effects [8]. In recent years, it has been found that DRSs also show activity against multiple human cancer types and, therefore, can be classified as Anti‐cancer peptides (ACPs) [8, 9]. DRSs have multiple anti‐tumour mechanisms [8, 10, 11], which can induce necrosis through cell membrane lysis, initiate apoptosis through mitochondrial membrane rupture, and some other non‐lytic membrane action modes [12, 13]. At the same time, DRSs can also enhance the efficacy of chemotherapy drugs and improve their sense of them [7]. In addition, DRSs themselves have specific tumour selectivity and less damage to normal tissues and cells [14]. This is because the phosphatidylserine and mucin expressed on the membrane surface of tumour cells make the membrane surface negatively charged, which makes it easier to bind to DRSs cationic peptides [15].

Dermaseptin‐PP is an α‐helix anticancer peptide first found in the skin secretions of Phyllomedusa palliata. In our preliminary study, we found Dermaseptin‐PP has a broad spectrum of antitumour activity. Compared with cisplatin and other commonly used clinical drugs, it has lower tissue toxicity, which has excellent potential for clinical application [16]. However, Dermaseptin‐PP and some other α‐helical anticancer peptides in the DRSs family still have some difficulties to overcome in clinical application—for example, haemolytic toxicity [17] because normal human red blood cells also have a small net negative charge [18]. Therefore, how to avoid the haemolytic toxicity of Dermaseptin‐PP and increase its accumulation in tumour sites is the key to giving full play to its antitumour effect. This study will provide a reliable reference for the antitumour application of Dermaseptin‐PP and other α‐helix members of the DRSs family.

Liposome is a kind of microvesicle with a lipid bilayer structure, which can enclose drugs in the bilayer to shield drug activity temporarily [19]. At the same time, it can also realise the targeted delivery and controlled release of drugs through structural modification, which has irreplaceable advantages in enhancing and attenuating drugs [20, 21]. At the site of tumour tissue, tumour cells undergo anaerobic respiration due to long‐term hypoxia, and the resulting lactic acid metabolism makes the tumour microenvironment appear weakly acidic [22]. In contrast, the average human tissue and blood microenvironment are neutral or weakly alkaline. Therefore, constructing a pH‐response liposome [23, 24] will limit the release of Dermaseptin‐PP in normal tissues. It is possible to avoid the risk of haemolysis and improve the anti‐tumour efficacy.

In this study, Dermaseptin‐PP will be loaded with pH‐sensitive membrane material DSPE‐Hyd‐PEG2k (Figure 1) to prepare a Der‐loaded‐pHSL with ph‐responsive properties. In DSPE‐Hyd‐PEG2k, the hydrazone bond [25] (‐Hyd‐) linking DSPE to PEG2k is a classical pH‐sensitive bond, which can be broken only under acidic conditions. Studies have shown that they will cleave 80% within 1 h at a pH below 6.5 [26]. That is, Der‐loaded‐pHSL can only dissociate and release Dermaseptin‐PP in the acidic environment of the tumour site and remains inactive in the normal tissue site until it is delivered to the tumour site and is dissociated or metabolised out of the body. This can effectively avoid the risk of haemolysis caused by Dermaseptin‐PP at high doses and improve the anti‐tumour effect of Dermaseptin‐PP, which will provide a new method and idea for the clinical application and tumour treatment of Dermaseptin‐PP.

FIGURE 1.

Structure of DSPE‐Hyd‐PEG_2k.

2. EXPERIMENTAL SECTION

2.1. Materials

Dermaseptin‐PP peptide (Der sequence for ALWKDMLKGIGKLAGKAALGAVKTLV‐NH₂; Synthesised by Shanghai Gill Biochemical Co., LTD.); 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine (DSPE, Xi'an Ruixi Company); DSPE‐Hyd‐PEG_2k (Xi'an Ruixi Company); Lecithin (SPC, Xi'an Ruixi Company); cholesterol (CHO, Xi'an Ruixi Company); Reagents used in cell experiments, such as CCK‐8, DAPI, and fetal bovine serum, were all obtained from Sigma Company.

2.2. Instruments

Electronic balance (BS224S, Sartorius, Germany); Laser particle size analyser (Nano‐ZS90, Malvern, UK); Rotary evaporator (R‐200, BUCHI, Switzerland); Constant temperature water bath oscillator (KW‐1000 DB, China Jintan Science and Technology Instrument Co., LTD.); Flow cytometry (Beckman Coulter, MoFlo XDP, USA); Small Animal Living Imager (MIIS, Molecular Devices, USA); High‐performance liquid chromatograpH (Shimadzu LC‐20ADXR Day Sshimadzu); C18 column (5 μm, 4.6 × 250 mm, Agilent, USA).

3. METHODS

3.1. Preparation of Dermaseptin‐PP liposomes

Dermaseptin‐PP liposomes were prepared according to the method of Ziyi Dong et al. [7]: Dermaseptin‐PP liposomes were prepared by membrane hydration. SPC, CHO, and DSPE‐Hyd‐PEG_2k (Figure 1) were weighed at a molar ratio of 65:25:10 (mol:mol:mol) and then mixed and dissolved in an appropriate amount of methylene chloride. Dichloromethane was removed to make a thin film using a rotary vapouriser, and 1 mL of 750 μg/mL aqueous Dermaseptin‐PP was added for vortex‐hydration. The probe was ultrasonic for 3 min (power 80 W, working time 5 s, intermittent 5 s). After ultrasonic, the solution was passed through 0.4, 0.2, and 0.1 μm polycarbonate membrane to prepare the pH‐sensitive Dermaseptin‐PP peptide liposome Der‐loaded‐pHSL. The non‐pH sensitive Dermaseptin‐PP peptide‐liposome Der‐loaded‐L was prepared using non‐pH sensitive DSPE‐mPEG_2k instead of pH‐sensitive DSPE‐Hyd‐PEG_2k. PEG‐SL and PEG‐pHSL blank control liposomes were prepared by the same method without the Dermaseptin‐PP peptide.

3.2. Particle size and potential

A Malvern particle size analyser determined the particle size and potential of Dermaseptin‐PP liposomes at 25°C. The results were averaged after three parallel operations.

3.3. Encapsulation rate and drug load

Dermaseptin‐PP aqueous solutions with concentrations of 1, 2, 4, 8, 12, 16, and 20 μg/mL were prepared. HPLC determined the linear range of Dermaseptin‐PP. The chromatographic conditions were as follows: Inertsil ODS‐3 column (250.0 × 4.6 mm, 5 μm); Mobile phase A: 0.1% TFA solution, mobile phase B: 0.1% acetonitrile solution; Mobile phase conditions: 65% A phase‐40% A phase in 25 min; Flow rate: 1 mL/min; Wavelength: 220 nm.

One hundred microlitres of Der‐loaded‐pHSL and Der‐loaded‐L solutions were absorbed, diluted with 900 μL acetonitrile, and mixed with vortexing for 10 min. The dilution was centrifuged at 12,000 r/min for 10 min, and the supernatant was separated. One millilitre of the supernatant was placed in a 10 mL volume‐volume flask, diluted in constant volume with deionised water, and shaken well to obtain the sample solution of Dermaseptin‐PP peptide liposome. The blank liposome reference solution was prepared by the same method. The content of Dermaseptin‐PP peptide in the sample solution was detected under the above chromatographic conditions. The drug's encapsulation rate (EE) and drug load (DL) were calculated according to the following formulas(1) and (2):

$$\mathrm{E}\mathrm{n}\mathrm{c}\mathrm{a}\mathrm{p}\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}(\mathrm{E}\mathrm{E}\%)=m_0/m_1\times 100\%$$ (1)

$$\mathrm{D}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}(\mathrm{D}\mathrm{L}\%)=m_0/m_1\times 100\%$$ (2)

m ₀: the weight of Dermaseptin‐PP encapsulated inside the liposomes; m ₁: total weight of Dermaseptin‐PP; m ₂: total weight of Dermaseptin‐PP and excipients.

3.4. Haemolysis test

The blood of healthy mice was prepared into 2% red blood cell suspension with PBS (pH 7.4), and 0.2–320 μg/mL Dermaseptin‐PP peptide liposome was added and incubated at 37°C for 3 h. The cell suspension was centrifuged at 5000 r/min for 10 min at 4°C. The supernatant was put into a 96‐well plate, and the UV absorbance of each supernatant was measured at 540 nm. PBS (pH 7.4) and 1% red blood cell lysate were used as negative and positive controls to calculate haemolysis:

$$\mathrm{H}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}=\left(A_s-A_n\right)/\left(A_p-A_n\right)\times 100\%$$

A _s is the absorbance value of the sample, A _p is the absorbance value of the positive control, and A _n is the absorbance of the negative control.

3.5. In vitro antitumour activity assay

CCK‐8 cytotoxicity assay was used to investigate the growth inhibition of Dermaseptin‐PP peptide‐modified liposomes on A549 cells. In our previous study, Dermaseptin‐PP was found to have potent cytotoxicity (Inhibition Rate > 90%) [16] on a variety of tumour cells at the concentration of 10‐5 M (~25 μg/mL). Therefore, we used 20 μg/mL as the highest concentration of the preparation for the cytotoxicity study.

A549 cells were cultured in DMEM medium (i.e. complete medium) containing 10% (v/v) FBS and 1% (v/v) penicillin‐streptomycin in a humidified incubator with 5% CO₂ at 37° C. Firstly, the tumour cells in the logarithmic growth phase were digested with 0.25% trypsin and suspended in single‐cell suspension with a complete medium. Then the cells were counted on a blood count plate. The cells were seeded at 8 × 103 cells/well/100 μL into 96‐well plates and incubated overnight. After washing with PBS, the cells were treated with 100 μL of Dermaseptin‐PP peptide liposome serum‐free medium solution with different concentrations (0.2, 2, 4, 10, 20 μg/mL), and incubated for 24 h. Then, 10 μL CCK‐8 reagent and 90 μL serum‐free medium were added to each well and incubated for 2 h. The absorbance value of each well was measured at 450 nm using a microplate reader, and the Inhibition Rate was calculated:

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{R}\mathrm{a}\mathrm{t}\mathrm{e}(\%)=1–A_{\mathrm{f}\mathrm{o}\mathrm{r}\mathrm{m}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{s}}/A_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$$

A _{formulations:} absorbance values of formulations holes; A _control: the absorbance value of the control hole.

3.6. Cellular uptake of liposomes at different pH conditions

Since the Dermaseptin‐PP peptide has no fluorescence, coumarin‐6 (C6) was used as a fluorescent probe to replace Dermaseptin‐PP to prepare pH‐sensitive C6‐loading‐pHSL and control C6‐loading‐L liposomes according to “2.1” method. The quantitative uptake of Dermaseptin‐PP peptide liposomes by A549 cells in different pH environments was studied by flow cytometry.

First, A549 cells were seeded in 6‐well plates at 3 × 10⁵ cells/mL density and incubated for 24 h. Cells were treated with C6 liposomes dissolved in different pH buffers (pH 5.0, 6.5, and 7.4) at 2 μg/mL and incubated for 4 h. After the culture medium was discarded and washed with PBS, the cells were digested with 0.25% trypsin and collected and centrifuged at 1000 rpm for 5 min. Cells were resuspended in 200 μL PBS, and fluorescence intensity was measured by flow cytometry. 1 × 10⁴ cells were recorded for each sample, and three re‐wells were set for each pH condition.

3.7. In vivo distribution

Female nude mice were purchased from Spefu (Beijing) Biotechnology Co., LTD. 8 × 10⁶ A549 cells were suspended in 100 μL medium and injected subcutaneously into the right abdomen of nude mice. Three A549 tumour‐bearing nude mice were injected with 200 μL of Free‐C6, C6‐Loader‐pHSL, and C6‐loader‐L solutions at 200 μg/mL (calculated as C6) through the tail vein respectively. After 2, 4, 8, 16, 24, 48, and 96 h of isoflurane anaesthesia, the images were taken in the IVIS living animal imaging system. After 96 h, each tumour‐bearing nude mouse's tumour tissues and normal organ tissues were collected for in vitro fluorescence imaging analysis.

3.8. Efficacy evaluation in vivo

To study the antitumour effect of Dermaseptin‐PP liposome (Der‐loaded‐L) in vivo in A549 tumour‐bearing mice. Tumour‐bearing mice with tumour volume of 100–150 mm³ were divided into five groups (n = 4), which were: (a) Der‐loaded‐pHSL (6 mg/kg); (b) Der‐loaded‐L (6 mg/kg); (c) Blank vector group (PEG‐pHSL); (d) Free Der; (e) PBS (pH 7.4) group. All groups were given drugs through the tail vein once every 3 days, a total of three times of administrations. The time of the first administration was defined as the first day, and mice's body weight and tumour size were recorded before each administration until the tumour volume of mice in the PBS group exceeded 1000 mm³. Digital vernier callipers measured tumour length (L) and width (W). Tumour volume (V) was calculated according to formula (1), and TGI (Tumour Growth Inhibition value) was further calculated according to formula (2):

$$V=\left(L\times W_2\right)/2$$ (1)

$$\mathrm{T}\mathrm{G}\mathrm{I}(\%)=\left(V_0-V_t\right)/V_0\times 100\%$$ (2)

V ₀ is the volume of tumour in PBS control group; V _t is the volume of tumour in experimental group.

3.9. Statistical methods

SPSS 22.0 software was used for statistical analysis. t‐test and one‐way analysis of variance was used for comparison between groups. p < 0.05 was considered statistically significant.

4. RESULTS AND DISCUSSION

4.1. Pharmacologic characterisation of liposomes

The particle size and particle size dispersion range of liposomes significantly impact liposomes' stability and encapsulation rate, which are the basis and critical links in the preparation process of liposomes [27]. The particle sizes of Der‐loaded‐pHSL and Der‐loaded‐L were (96.78 ± 1.50) nm and (90.62 ± 1.50) nm, respectively, which met the general particle size requirements of liposomes (<200 nm) (Figure 2). PDI was 0.15 ± 0.02 and 0.12 ± 0.03, respectively, indicating that the size distribution of liposomes was uniform. Zeta potential is the total charge obtained by liposomes in the medium. The larger its absolute value, the greater the electrostatic repulsion between liposomes and the more stable the system [28]. The Zeta potentials of Der‐loaded‐pHSL and Der‐loaded‐L were (6.94 ± 0.58) mV and (−17.6 ± 1.70) mV, respectively, indicating that both of them could avoid aggregation by surface charge repulsion. The encapsulation rate and drug load are directly related to the therapeutic dose of liposome drugs. The larger the value, the more likely it is to meet the clinical requirements [17]. The encapsulation rate and drug load of Der‐loaded‐pHSL were 86.26 ± 0.19 and 7.15 ± 0.01 respectively. The encapsulation rate and drug load of Der‐loaded‐L were 97.81 ± 0.05 and 7.10 ± 0.10, respectively, which met the pharmacologic standards (Table 1).

FIGURE 2.

Nanoparticle size of lipidosome (Mean ± SD, n = 3).

TABLE 1.

Characterisation of lipidosome (Mean ± SD, n = 3).

Nanoparticle	Size (nm)	PDI	Zeta (mV)	EE (%)	DL (%)
Der‐loaded‐pHSL	96.78 ± 1.50	0.15 ± 0.02	6.94 ± 0.58	86.26 ± 0.19	7.15 ± 0.01
Der‐loaded‐L	90.62 ± 1.50	0.12 ± 0.03	−17.6 ± 1.70	97.81 ± 0.05	7.10 ± 0.10

4.2. Haemolytic

As a particular type of cell, red blood cells perform various bodily functions, such as energy metabolism, material transport, and signal transmission [29]. Many antimicrobial peptides with α‐helical structure, such as Dermaseptin‐PP peptide, can change the permeability of erythrocytes to intracellular cations and eventually lead to cell lysis [30]. It was found that Dermaseptin‐PP had a measure of haemolytic toxicity. The haemolysis rate of Dermaseptin‐PP at 320 μg/mL was as high as 90.43%. The haemolysis rate of Dermaseptin‐PP was significantly dose‐dependent in the range of 20 ~ 320 μg/mL. However, at 160 μg/mL concentration, the haemolysis rate of Der‐loaded‐pHSL was reduced by about 40% compared with that of Free Der. At the concentration of 320 μg/mL, the haemolysis rate of Der‐Loaded‐L was reduced by about 60% compared with that of Free Der (Figure 3). This result indicated that the preparation of Dermaseptin‐PP as liposomes could significantly reduce the haemolysis toxicity, which provided a possibility for its administration in vivo.

FIGURE 3.

The relationship between Dermaseptin‐PP concentration and haemolysis.

4.3. Antitumour activity in vitro

A549 human non‐small cell lung cancer cells were used as a tumour cell model to investigate the antitumour activity of Dermaseptin‐PP peptide‐modified liposomes in vitro. The results showed that free Der, Der‐loaded‐pHSL, and Der‐loaded‐L had dose‐dependent cytotoxicity to A549 cells in the concentration range of 2–20 μg/mL. As mentioned in the introduction, the mechanism of Der cytotoxicity can be divided into two types: one is to act on the cell membrane to form pores and cause cell lysis and death; the other is to enter the cell to induce cell apoptosis. At low concentrations of Der (2–4 μg/mL), the non‐membrane‐breaking mechanism was dominant, and the cytotoxicity was mainly generated by the part of Der that entered the cell. Compared with free Der, Der‐loaded‐pHSL has more significant antitumour activity, which may be because liposomes can more easily enter cells through the endocytic pathway by utilising the characteristics of cell membrane fusion [31]. At a high concentration of Der (>10 μg/mL), the mechanism of membrane breaking was dominant, and the cytotoxicity was mainly caused by the direct interaction between Der and the cell membrane. Since Der‐loaded‐L is non‐pH‐responsive, some Der is still blocked inside the liposome, and cytotoxicity is only generated by the fraction that enters the cell. However, free Der could directly interact with the cell membrane, so with the increase in concentration, the cytotoxicity of Der gradually exceeded that of Der‐loaded‐L. Der‐loaded‐PHSL is pH‐responsive and can dissociate in the acidic environment of the tumour site. Der entering the cell can induce apoptosis by interfering with the cell cycle, and Der retained outside the cell can play a killing role through the membrane‐breaking mechanism, so it has stronger cytotoxicity (Figure 4).

FIGURE 4.

Dose‐dependent cytotoxicity of Dermaseptin‐PP liposome.

4.4. Cellular uptake of liposomes at different pH environments

In the above studies, we found that Der‐loaded‐pHSL has potent cytotoxicity, which the acidic environment of tumour cells may cause. Therefore, we further studied the uptake of Der‐loaded‐pHSL by A549 cells in different pH environments. We found that the cellular uptake of Der‐loaded‐pHSL increased with the decrease in pH, and the cellular uptake of Der‐loaded‐pHSL at pH 5 was nearly 2 times higher than that at pH 7.4. Moreover, in the acidic environment of pH 6.5 and 5.0, the cell uptake of Der loader‐pHSL was significantly higher than that of Der Loader‐L (Figure 5). These results verified our hypothesis that the acidic tumour microenvironment was conducive to tumour cells' uptake of Der Loader‐pHSL to exert drug effect better. In addition, we found that pH 5.0 increased the uptake of Der‐Loaded‐L. Since Der‐loaded‐L has a high negative Zeta potential (−17.6 ± 1.70 mV) and negative charge on the tumour cell membrane surface, we speculate that this result is because H+ in an acidic environment reduces the charge repulsion between Der‐loaded‐L and cell membrane. Furthermore, promotes cell absorption to a certain extent [32]. Similarly, the uptake of C6‐Loaded pHSL was higher than that of C6‐Loaded‐L at pH 7.4. We speculated that this was because OH‐ in the alkaline environment reduced the charge repulsion between the positively charged C6‐loaded‐pHSL and the tumour cell membrane. This promotes its uptake by tumour cells.

FIGURE 5.

Cellular uptake of Dermaseptin‐PP peptide liposomes at different pH environments (Significant differences were indicated by p values, *p < 0.05, **p < 0.01 and ***p < 0.001 compared with pH 7.4 group; ^# p < 0.05, ^### p < 0.001, C6‐loaded‐pHSL vs. C6‐loaded‐L, at same pH).

4.5. In vivo distribution

Based on the conclusions obtained from cell experiments, we further used the tumour‐bearing nude mouse model of A549 cells to explore the distribution characteristics of Der‐loaded‐pHSL in vivo. We found that C6‐loaded‐pHSL and C6‐loaded‐L could accumulate at the tumour site compared with free C6, indicating that Liposomal nanoparticles have specific tumour‐targeting effects, which may be achieved through the EPR effect. It is worth noting that in the imaging images at each time point after 4 h, the fluorescence signal of the tumour site in the C6‐loader‐pHSL group was significantly higher than that in the C6‐loader‐L group. This difference is more evident in the fluorescence imaging of isolated tumour tissues after 96 h, which indicates that C6‐loader‐pHSL with pH‐responsive structure is more likely to accumulate in the tumour site and has a more substantial targeting effect compared with ordinary liposomes (Figure 6).

FIGURE 6.

Distribution characteristics of liposome preparations in vivo.

4.6. Efficacy evaluation in vivo

These studies indicated that our pH‐sensitive liposomes could significantly reduce the haemolytic toxicity caused by Dermaseptin‐PP and have a significant tumour‐targeting effect. Based on this, we further investigated the antitumour activity of Der‐loaded‐pHSL in vivo using A549 tumour‐bearing nude mouse model. The results showed that the tumour inhibition rates of Der‐loaded‐pHSL and Der‐loaded‐L were significantly higher than those of free Der, which indicated that Dermaseptin‐PP could significantly enhance its antitumour effect after being loaded with liposomes (Figure 7). In addition, the tumour inhibition rate of Der‐loaded‐pHSL was significantly higher than that of Der‐loaded‐L and pHSL, which indicated that the construction of pH‐sensitive liposomes coated with Dermaseptin‐PP had more significant antitumour advantages compared with ordinary liposomes.

FIGURE 7.

Evaluation of the antitumour effect of Der‐loaded‐pHSL (*p < 0.05 and **p < 0.01 compared with free Der group).

5. CONCLUSIONS

Dermaseptin‐PP is a promising α‐helix anticancer peptide, but its haemolytic toxicity limits its further application. In this study, the characteristics of tumour microenvironment‐responsive liposomes were fully exploited, and Dermaseptin‐PP peptide with a unique tumour‐killing mechanism was encapsulated in the body cavity. Der‐loaded‐pHSL significantly reduces the haemolytic toxicity of Dermaseptin‐PP and achieves tumour targeting by utilising the EPR effect and pH responsiveness of liposomes themselves. Therefore, the Der‐loaded‐pHSL nanoliposome designed in this study is a novel anticancer agent with great potential, which is expected to provide new ideas and methods for tumour treatment.

AUTHOR CONTRIBUTIONS

Changhai Wang: Formal Analysis; Methodology; Writing ‐ Original Draft. Ziyi Dong: Data Curation; Investigation. Qing Zhang: Writing Review & Editing. Mingxue Guo: Validation. Wenjun Hu: Validation. Shuang Dong: Data Curation; Investigation. Tangthianchaichana Jakkree: Validation. Yang Lu: Funding Acquisition; Conceptualisation. Shouying Du: Conceptualisation; Resources.

CONFLICTS OF INTEREST STATEMENT

The authors declare that they have no competing interests.

PERMISSION TO REPRODUCE MATERIALS FROM OTHER SOURCES

None.

Wang, C. , et al.: Stimulus‐responsive nano lipidosome for enhancing the anti‐tumour effect of a novel peptide Dermaseptin‐PP. IET Nanobiotechnol. 17(4), 352–359 (2023). 10.1049/nbt2.12128

DATA AVAILABILITY STATEMENT

Data generated during the study are subject to a data sharing mandate and available in a public repository that does not issue datasets with DOIs.