Introduction

Hepatocellular carcinoma (HCC) is known as a primary liver tumor and accounts for more than 90% of primary liver tumors. HCC is diagnosed in approximately 85% of patients with cirrhosis (Grandhi et al. 2016). This cancer is currently the fifth most common cause of cancer worldwide and the second leading cause of cancer death in men after lung cancer (Sayiner et al. 2019). The life expectancy of patients with HCC depends on the stage of the cancer at diagnosis. In the early stages, treatments such as surgical resection, liver transplantation, and local ablation can improve patient survival. However, the disease is usually diagnosed in advanced stages where these treatments are ineffective (Tunissiolli et al. 2017). The most important treatment solution is the use of chemotherapy, which does not always have satisfactory effects and is accompanied by side effects (Yang et al. 2024). In addition, drug resistance can also lead to failure of treatment (Ladd et al. 2024). Among the methods of interest to deal with these limitations is the use of drug nanocarriers for targeted drug delivery, which with the targeted release of the drug leads to a reduction in the dosage, side effects and more effective effect of the drug. In addition, nanocarriers can protect the drug from degradation in the internal environment of the body and deliver cancer drugs that have low solubility in water to the tumor site without damaging the surrounding tissues (Yang et al. 2022; Ji et al. 2024). Generally, studies have shown that cellular junctions on the surface of blood vessels of healthy tissues prevent the passage and absorption of molecules larger than 2–4 nm (Jiménez et al. 2014). As a result, nanoparticles (NPs) cannot penetrate them and continue to circulate in the blood and are finally excreted, while cellular junctions in the blood vessels of tumors are weak due to the desire to absorb nutrients, and macromolecules with the size of 600 nm can pass through them. On the other hand, NPs that penetrate the cancerous tissue cannot be removed because tumors lack an effective lymphatic system (Martin and Jiang 2009; Roux and Gavard 2019). This process is based on the enhanced permeation and retention (EPR) effect, which leads to the accumulation of NPs within solid tumors. In the first generation of NPs-based drug delivery, since angiogenesis is intensified in the tumor site, drug delivery is usually based on EPR as the passive method, but this method is not accurate and can still lead to complications for healthy tissues (Wu 2021; Sharifi et al. 2022). For this reason, in recent years, drug delivery by targeted NPs has received more attention as the active method. Active targeting involves the delivery of the drug to the target site using ligands that will bind to specific receptors that are overexpressed on target cells, which potentially leads to fewer side effects and improved drug tolerance (Li et al. 2023). It has been shown that after active absorption of NPs, drug-carrying NPs cross the cell membrane through endocytosis or endocytosis-independent pathways such as insertion and diffusion through lipid bilayers (Li et al. 2017; Ju et al. 2020). Accordingly, the tendency of nanoparticles to accumulate in tumor sites can be increased by using specific ligands for surface receptors on cancer cells that are more expressed in these types of cells (Hong, Li et al. 2023). SERPINB3 (SB3) is one of the antigens which is undetectable in normal hepatocytes, but their expression progressively increases in chronic liver diseases, dysplastic nodules, and HCC, therefore is considered as a specific marker of HCC (Pontisso 2014). SB3 (formerly known as squamous cell carcinoma antigen-1 or SCCA1) is a member of the family of serine-protease inhibitors, which protects cells from oxidative stress conditions, but in chronic liver damage, this serpin may lead to HCC through different strategies, including induction of epithelial to mesenchymal transition, cell proliferation and invasiveness, inhibition of apoptosis, and decrease of desmosomal junctions (Quarta et al. 2010; Pontisso 2014). In addition, SB3 may also contribute to tumor cell resistance to anti-neoplastic drugs through its binding to the respiratory Complex I and protect cancer cells from the pro-oxidant activity of chemotherapeutic agents (Ciscato et al. 2014). Studies have shown that SB3 acts as a hepatitis B virus (HBV) receptor, and a key recognition element in this process is the PreS1 (21–47 amino acids) peptide, which is part of one of the viral envelope proteins (De Falco et al. 2001). Therefore, this peptide can be used as a potential targeting agent against HCC cells. In this regard, Jha et al. confirmed the specific binding and uptake of PreS1 peptide-functionalized gold nanoparticles (Au NPs) into HCC cells (Jha et al. 2017).

For targeted delivery and among the nanocarriers, polymeric nanoparticles (PNPs) are as suitable carriers that have extensive applications in drug delivery systems (Taghipour-Sabzevar et al. 2019). Chitosan (CS) is a linear polysaccharide which widely used as a suitable bio-nanocarrier for drug delivery because of its biocompatibility, biodegradability, and modifiability (Herdiana et al. 2022). Also, CS is the only polycation in nature whose solubility and charge density are affected by pH and acetylation level. The new or improved chitosan properties can be obtained by adding different functional groups or macromolecular components into the chitosan structure. These modifications enable it to be made in a variety of sizes and structures (Ma et al. 2020; Wang and Zhuang 2022). On the other hand, in recent years, many studies have been conducted on alternative chemotherapy drugs to be more effective with fewer side effects. Significant studies showed that antimicrobial peptides (AMPs) have anticancer properties. Among AMPs, Cationic peptides have received more attention (Moravej et al. 2018). Generally, cationic antimicrobial peptides (CAPs) are a promising new class of natural-source drugs that may avoid the deficiency of conventional chemotherapy because certain CAPs exhibit cytotoxicity against a broad spectrum of cells, especially cancer cells (Tornesello et al. 2020). CAPs with a positive charge have a greater tendency to interact with the membrane of cancer cells, which are more negative than normal cells. However, CAPs act on eukaryotic cells non-selectively; therefore, to reduce the cytotoxic effects on normal cells, it is suggested that cell-specific release systems better be used for the targeted release of these peptides (Hoskin and Ramamoorthy 2008). CM11 is a cationic antimicrobial peptide that consists of 11 amino acids. Based on studies, the anticancer property of the CM11 has also been proven (Taghipour-Sabzevar et al. 2020). It has been shown that cationic peptides such as CM11 through processes including pore-forming in the cell membrane, blocking the formation of some extracellular structures, and interfering with essential intracellular activities like protein synthesis can induce cell death (Mohammadi Azad et al. 2017; Mirnejad et al. 2023). Since these peptides are synthetic and their production may create economic limitations, and on the other hand, there are some possible side effects, so reducing the effective dose can lead to the optimal use of these peptides in the clinic. One of the methods that can be used to achieve this goal is to use it simultaneously with another drug, which leads to a reduction in dosage and even better effectiveness in a synergistic way (Shen et al. 2017). Curcumin (Cur) is a polyphenol isolated from the rhizomes of Curcuma longa and has been found to possess a variety of biological properties such as immunomodulatory, anti-inflammatory, antioxidant, neuroprotective, and anticancer. Curcumin acts on the regulation of different aspects of cancer development, including initiation, metastasis, angiogenesis, and progression (Tomeh et al. 2019). Findings have shown that the anticancer properties of curcumin are related to its diverse interactions with key molecular pathways related to various biological processes, such as proliferation, apoptosis, metastasis, angiogenesis, and inflammation (Sultana et al. 2021). In addition, it has been found that curcumin can specifically target cancer stem cells. These cells are mainly responsible for resistance to chemotherapy treatments and relapse in cancer patients (Zang et al. 2014).

Based on the description provided, in this study, the targeted release of curcumin (Cur) and CM11 peptide alone and together against hepatocellular carcinoma was evaluated using chitosan nanoparticles coated with Pres1 that target the SB3 antigen of HCC cells.

Materials and methods

Materials

CM11 (Mw = 1414.87 Da, Seq = WKLFKKILKVL) and PreS1 (Mw = 3133.3 Da, Seq = CPNFDWDPNNSNAGFAPDLQHDPFFGLP) peptides were purchased from Synpeptide Co.,Ltd (Shanghai, China). CS with deacetylation degree of 85% and medium molecular weight (Mw = 90–310 kDa), Tripolyphosphate (TPP), dimethyl sulfoxide (DMSO), and 3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (USA). 1-Ethyl − 3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxy sulfosuccinimide (NHS) and glacial acetic acid were purchased from Merck (Germany). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (USA). HepG2 (Human liver cancer cell line), A549 (Human lung cancer cell line), and Hu02 (Human normal fibroblast) cells were obtained from the National Center of Genetic and Biological Resources of Iran.

Synthesis of CS NPs

The preparation of CS-NPs was done almost according to the ionic gelation method described by Calvo et al. (Calvo et al. 1997). First, 1.16 mg of CS, which has been optimized according to the previous works of the group (Taghipour-Sabzevar et al. 2020), was added to 1% v/v aqueous acetic acid and stirred for 1 day. Then TPP was dissolved in deionized water, and a TPP solution with a concentration of 1 mg/ml was prepared. The TPP solution was added drop by drop to the CS solution while stirring until it became cloudy, and then it was stirred for 2 h at room temperature. The above steps were repeated to prepare CS NPs containing single and dual drugs. Before adding the TPP solution, Cur and CM11 were added one by one and at the same time to the CS mixing solution, and after it became uniform, the TPP solution was added as before (Fig. 1a). Finally, the samples were centrifuged at 15,000 rpm and 4 °C for 25 min, and CS NPs free of drugs and containing drugs were separated, and the supernatant was collected to check the drug loading efficiency.

Fig. 1
figure 1

Preparation of chitosan nanoparticles containing Cur and CM11 peptide by ionic gelation method (a) and coating reaction of chitosan nanoparticles with PreS1 peptide using EDC and NHS (b)

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CS NPs coating with PreS1 peptide

EDC and NHS were used to activate the carboxyl group of the PreS1 peptide and connect it to the amine group of CS (Fig. 1b). First, the NPs were dispersed in distilled water, then PreS1 was dissolved in distilled water, then 100 µl of NHS (0.1 M) and 25 µl of EDC (0.4 M) (Bartczak and Kanaras 2011) were added to 100 µl PreS1 with a concentration of 1 mg/ml, and they were stirred for 1 h at room temperature to activate the functional groups. Subsequently, drop by drop was added to the dispersed CS-NPs, mixed once more for an hour at room temperature, and then centrifuged for 30 min at 15,000 rpm to eliminate unreactive material. The coated NPs (PreS1-drug-CS NPs) were then collected.

Characterization of the NPs

Size and zeta potential

At a temperature of 25 °C, the size of the produced NPs was measured using a dynamic light scattering (DLS) device. Using this method, the size of the NPs was measured at 258 nm wavelength and 1.35 refractive index after they were initially diluted with water to a concentration of 0.5 mg/ml. Additionally, the ZetaCheck device from the Nanoflex company was used to measure the zeta potential of nanoparticle suspension in water; the device’s output was limited to a numerical display.

Morphology

The morphology of the NPs was examined using scanning electron microscopy (FE-SEM) (Philips, Nederland). Following dilution, the NPs underwent ultrasonication and were vacuum-dried in an oven for eight hours. In the following, a tiny coating of gold was applied, and imaging was carried out.

Stability

PreS1-Cur-CM11-CS NPs were dispersed in distilled water, 5% glucose, and 0.9% NaCl, and then maintained in an incubator to assess the physical stability of the particles under physiological conditions. After three weeks, PreS1-Cur-CM11-CS NPs’ hydrodynamic diameter and zeta potential were measured at various periods using DLS and Zeta sizer (Abolhassani et al. 2020).

Fourier-transform infrared spectroscopy (FTIR)

The coating of CS NPs by Pres1 peptide was verified using an FTIR test (Perkin Elmer Co., USA). The 1–3 mg powder samples were combined with KBr and formed into tablets at 1000 psi pressure, and then the FTIR of the samples was obtained in the range of 400–4000 cm− 1.

Drug loading

To load drugs, Cur and CM11 were added to the CS solution at a weight ratio of 1:3 and 1:2 (drug: CS) for curcumin and CM11, respectively. Drop by drop, TPP solution was added to the drug-containing CS solution after CUR and CM11 had been added to it individually and together. This resulted in the drugs being trapped inside the CS-NPs. The solution with the NPs was centrifuged for 20 min at 14,000 rpm to collect the CS-NPs and verify the drug loading. To ensure that there were no unloaded drugs left, they were washed three times more. Subsequently, the CS nanoparticle supernatant was collected in both mono- and dual-drug modes. A spectrophotometer was used to measure the amounts of unloaded drug at 280 nm for the CM11 and 425 nm for the Cur, with the results being compared to standard plots. Formulas 1 and 2 were used to determine the drug loading content (LC) and drug loading efficiency (LE) of loaded CUR and CM11 (Rahmani et al. 2023).

$${\rm{LC}} = {\matrix{{\rm{Total}}\>{\rm{weight}}\>{\rm{of}}\>{\rm{the}}\>{\rm{drug}}\> \hfill \cr {\rm{ - }}\>{\rm{Weight}}\>{\rm{of}}\>{\rm{the}}\>{\rm{free}}\>{\rm{drug}}\>{\rm{remains}} \hfill \cr} \over \matrix{{\rm{Total}}\>{\rm{weight}}\>{\rm{of}}\>{\rm{drug}}\> \hfill \cr {\rm{containing}}\>{\rm{nanoparticle}} \hfill \cr} } \times \>100$$
(1)
$${\rm{LE}} = {\matrix{{\rm{Total}}\>{\rm{weight}}\>{\rm{of}}\>{\rm{the}}\>{\rm{drug}}\> \hfill \cr {\rm{ - }}\>{\rm{Weight}}\>{\rm{of}}\>{\rm{the}}\>{\rm{free}}\>{\rm{drug}}\>{\rm{remains}}\> \hfill \cr} \over {{\rm{Total}}\>{\rm{weight}}\>{\rm{of}}\>{\rm{the}}\>{\rm{drug}}}} \times \>100$$
(2)

Drug release profile

Placed inside the dialysis bag (MWCO: 8 kDa) were Pres1-Cur-CM11-CS NPs, Cur-CM11-CS NPs, and free solutions of the Cur and CM11 (1 mg/ml). These were then placed in 20 CC of PBS (pH 7.4), and ethyl acetate buffer (pH 5.5), and incubated at 37 °C for 48 h. The releasing media were made with 0.5% (w/v) sodium dodecyl sulfate (SDS) since Cur was not soluble in the aqueous medium (Phillips et al. 2012). 1 CC sample was extracted from the release medium at the designated intervals of 0.5, 1, 2, 4, 8, 12, 24, and 48 h in order to verify the drug release. UV spectroscopy was used to determine the concentration of the released drug in the medium. A fresh medium volume of 1 CC was added to the solution in order to uphold the law of conservation of mass. Relationships 3 and 4 were then used to compute the cumulative release of Cur and CM11.

$$\eqalign{& {\rm{The}}\>{\rm{drug}}\>{\rm{is}}\>{\rm{released}}\>\left( {{\rm{mg}}} \right) \cr & = {\rm{Concentration}} \cr & \times \>{\rm{The}}\>{\rm{volume}}\>{\rm{of}}\>{\rm{the}}\>{\rm{release}}\>{\rm{medium}} \cr & \times \>{\rm{Dilution}}\>{\rm{coefficient}} \cr} $$
(3)
$$\eqalign{\>{\rm{Cumulative}}\>{\rm{release}}\>{\rm{percentage}}\> = & \>{{{\rm{Sampling}}\>{\rm{volume}}} \over {{\rm{Volume}}\>{\rm{of}}\>{\rm{the}}\>{\rm{release}}\>{\rm{medium}}\>}} \cr & \times \>\>{\rm{P}}({\rm{t}} - 1)\> + \>{\rm{Pt}} \cr} $$
(4)

Pt = Cumulative release rate at time t.

P(t-1) = Cumulative release rate in the previous t time.

In vitro cytotoxicity assessment (MTT assay)

HepG2 cell lines as a hepatocellular carcinoma cell line, along with A549 (lung cancer cell line, which lack SB3 antigen) and Hu02 (Human normal fibroblast cell line) as controls were used to measure the toxicity of NPs. Cells were cultured in T25 flasks containing DMEM culture medium with 10% (v/v) fetal bovine serum (FBS); this medium is called complete medium. The cell flask was incubated at 37 °C and contained 5% CO2 gas. The number of cells was counted by hemocytometer slide, subsequently, 96-well plates were seeded with 100 µl of a medium containing around 1 × 104 cells, and the cells were cultured overnight. After removing the media, three repetitions of treatment groups with varying doses of CM11/Cur (ranging from 3/1.9 µg/ml to 24/15.2 µg/ml) were added to the wells and incubated for 24 and 48 h. Groups were treated with non-coated and coated NPs containing drugs (single and dual), and free drugs including Pres1-Cur-CM11-CS NPs, Pres1-Cur-CS NPs, Pres1-CM11-CS NPs, Cur-CM11-CS NPs, Cur-CS NPs, CM11-CS NPs, CS NPs, free Cur, and free CM11. Subsequently, PBS was used to wash the medium in the wells. Then the medium in the wells was drained and washed with PBS. In a dark environment, 100 µl of 0.5 mg/ml MTT solution was added to the wells, and they were incubated for 4 hours. After 4 hours, the formazan crystals were dissolved by adding 100 µl of DMSO solvent after removing the MTT solution. The purple formazan dye solution’s optical absorbance was measured at 540 nm in an ELISA reader once the crystals had dissolved and the wells’ color had become apparent (Taghipour-Sabzevar et al. 2020).

Assessment of apoptosis by flow cytometry

An annexin V-FITC/PI staining kit was used to examine the necrosis and apoptosis that PreS1-Cur-CM11-CS NPs caused in the HepG2 cell line. Briefly, first, 3 × 105 HepG2 cells were seeded in a 24-well plate, and after one day, the cells stick to the bottom of the plate. Then HepG2 cells were treated with control groups, free CS NPs, and PreS1-Cur-CM11-CS NPs with concentrations of 3, 6, 12, and 24 µg/ml. After 24 h, the cells were detached from the bottom of the plate, and after washing three times with PBS, PBS containing 2% fetal bovine serum was used to transfer the cells from the laboratory to the place of the flow cytometry device. After that, a buffer containing 2.5 µl of Annexin and 5 µl of PI was combined with each of the treated samples, and the mixture was gently stirred. The samples were heated for 30 min in the dark, and then flow cytometry was used to read them (Taghipour-Sabzevar et al. 2020).

Statistical analysis

GraphPad Prism software is used to report the results as mean ± standard deviation (SD). p ≤ 0.05 was considered to be statistically significant.

Results and discussion

Characterization of CS NPs and Pres1-coatd CS NPs

According to the previous study, the optimal concentration of CS solution was 1.16 mg/ml, and TPP solution was added at a ratio of 1:3 (TPP: CS solution) to CS solution to prepare NPs (Taghipour-Sabzevar et al. 2020). The size and size distribution of CS NPs were measured by the DLS device, which is shown in Table 1; Fig. 2a. After adding Cur and CM11 individually and collectively to the CS NPs, nanoparticle size was measured again, as shown in Table 1, the addition of Cur and CM11 to nanoparticles increases their size, so the simultaneous addition of two drugs increases the average size of nanoparticles from 45 nm (CS NPs) to 95 nm (Cur-CM11-CS NPs), respectively, which have good potential for passive drug delivery and release of the drug at the tumor site by EPR effect. In addition, the morphology and size of Cur-CM11-CS NPs were analyzed by SEM which is presented in Fig. 2b. Based on SEM analysis, the Cur-CM11-CS NPs size was approximately 105 nm, which was matched with the results obtained from the DLS. Also, SEM showed that the proportions used in the synthesis of nanoparticles were appropriate and prevented the aggregation of nanoparticles. The morphology of NPs was also uniform and spherical.

Fig. 2
figure 2

Size distribution (a) and SEM image (b) of Cur-CM11-CS NPs

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Table 1 Mean size and polydispersity index (PDI) of CS NPs, Cur-CS NPs, CM11-Cs NPs, Cur-CM11-CS NPs, Pres1-Cur-CM11-CS NPs, as well as, percentage of drug loading content (LC%), and drug loading efficiency (LE%) for cur and CM11 drugs
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Fig. 3
figure 3

Size distribution (a) and SEM image (b) of PreS1-Cur-CM11-CS NPs

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In the following, to active targeting and greater effectiveness of the drug delivery system, PreS1 peptide was added as an active targeting agent to the surface of the CS NPs. After binding PreS1 with a ratio of 1:10 (PreS1: CS) to CS NPs, the size and size distribution of Pres1-Cur-CM11-CS NPs were measured by the DLS, which is shown in Table 1; Fig. 3a. In addition, the shape and size of Pres1-Cur-CM11-CS NPs were analyzed and confirmed by the SEM, as shown in Fig. 3b. Based on DLS measurement, the average size of Pres1-Cur-CM11-CS NPs was 132 nm. According to the SEM imaging, the NPs were spherical and homogenous and had a size of about 120 to 145 nm, which is very close to the results obtained by DLS. The shape and size of nanoparticles affect how they are seen by body cells and thus determine their distribution, toxicity, uptake, and targeting ability (Ankamwar 2012; Dolai et al. 2021). For example, it has been reported that 100 nm particles exhibited a 2.5-fold greater uptake compared to 1 μm particles and a 6-fold greater uptake than 10 μm particles (Rizvi and Saleh 2018). Many reports confirmed that the size of the nanoparticle determines biological fate. Generally, as the particle size decreases, their surface area to volume ratio increases. This means that more of the drug is closer to the surface of the particle, which results in faster drug release. In addition, it has been shown that particles with a size of 200 nm or larger tend to activate the lymphatic system and are therefore more quickly removed from the blood circulation. Based on many evaluations, the optimal size for a nanoparticle is approximately 100 to 200 nm (Buzea et al. 2007; Rizvi and Saleh 2018). Nanoparticles with this range of sizes can be a suitable option for a drug delivery system by crossing the BBB, delivering a sufficient amount of drug due to the high surface-to-volume ratio, and avoiding immediate clearance by the lymphatic system (Buzea et al. 2007). The findings of this study showed that Pres1-Cur-CM11-CS NPs have an average size of 132 nm, which based on the topics discussed above, is a suitable size to be used as a drug delivery system. However, although the small size has advantages, it can lead to the aggregation of nanoparticles due to the large surface area, but this phenomenon can be controlled through the surface charge (zeta potential) of nanoparticles (Honary and Zahir 2013a, b). The zeta potential (ZP) of nanoparticles is among other important factors that, in addition to controlling this phenomenon, can affect the fate and effective use of NPs via the determination of the blood circulation time, uptake rate, and the intended target cells. In the bloodstream, conventional nanoparticles and negatively charged particles can be rapidly opsonized and massively cleared by macrophages (Honary and Zahir 2013a, b). On the other hand, zeta potential along with the size and shape of the nanoparticles play a key role in the cellular uptake of NPs. Uptake can be viewed as a two-step process: first, a binding step on the cell membrane and second, the internalization step. It seems that the attachment of nanoparticles to the cell membrane is mostly influenced by the surface charge of the particles (Lesniak et al. 2013; Behzadi et al. 2017). Nanoparticles with a higher surface charge (mostly positive charge) have a greater binding tendency to the cell membrane, which has a dominant negative charge (typically − 40 to -80 mV), and show higher cellular uptake (Sabourian et al. 2020). What facilitates and intensifies this uptake is the electrostatic interactions between the anionic cell membrane and the cationic nanoparticles. It should be noted that compared to normal cells, the surface of cancer cells is usually more negative due to the translocation of negatively charged constituents of the inner layer of the cell membrane (e.g., phosphatidylserine) to the cell surfaces (Honary and Zahir 2013a, b; Klähn and Zacharias 2013; Wodlej et al. 2019). Accordingly, in this study, the zeta potential of CS NPs was + 57.8, and after coating with PreS1 in a ratio of 1:10 (PreS1: CS), it decreased to + 27.6 due to the negative charge of PreS1 with net charge − 4 at pH 7 and Iso-electric point at pH 3.3. According to the size and zeta potential results, it can be claimed that Pres1-Cur-CM11-CS NPs have the appropriate intrinsic standards as a drug release system. It is noteworthy that the aggregation of the NPs is mainly due to the van der Waals force (Shrestha et al. 2020). A larger zeta potential indicates more charge of the NPs, which thereby results in a larger Debye length making the NPs not sense the van der Waals force, therefore, the aggregation is avoided especially in relation to particles with the size obtained in this study (Honary and Zahir 2013a, b). In general, particles will reach an established dispersion when the absolute value of ZP is above ± 30 mV due to the electric repulsion between particles (Honary and Zahir 2013a, b; Teo et al. 2022). Accordingly, the ZP of the Pres1-Cur-CM11-CS NPs can be concluded that the possibility of particle aggregation is very low. In addition to these characteristics, the polydispersity index (PDI) as a parameter which shows the suitability of the NP formulation was assessed (Danaei et al. 2018; Herdiana et al. 2022). PDI is basically a representation of the distribution of size populations within a sample. The numerical value of PDI ranges is from 0.0 to 1.0, with 0.0 representing a perfectly uniform sample concerning the particle size while 1.0 has a highly polydisperse sample with multiple particle size populations (Danaei et al. 2018). In this study, the PDI for nanoparticles containing a single drug (peptide or Cur) was below 0.2, while for nanoparticles containing two drugs (peptide + Cur) and coated (PreS1-CS NPs), was below 0.25. Generally, values of 0.2 and below are most commonly deemed acceptable in practice for polymer-based nanoparticles. It is considered that the CS NPs-based delivery system has an adequate homogeneity. Of course, according to the results of this study, it should be noted that the delivery system has sufficient homogeneity.

Fig. 4
figure 4

FTIR spectra of Cs NP, PreS1 peptide, and PreS1-Cs NPs

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Fig. 5
figure 5

Cumulative release of Cur and CM11 from Pres1-Cur-CM11-Cs NPs and Cur-CM11-Cs NPs at pH 7.4 (a) and pH 5.5 (b)

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FTIR of coated PreS1-CS NPs

In the presence of EDC as an activator of amine and carboxyl groups, the carboxylic group of PreS1 peptide coupled to amino groups of chitosan and covered the chitosan nanoparticles. To analyze the linkage between the PreS1 peptide and chitosan nanoparticles, a FTIR test was performed for the PreS1 peptide, CS NPs, and PreS1-CS NPs. Figure 4 represents the results of FTIR analyses. The third type amide group, the second type amide, and the first type amide are represented by the peaks 1308 cm− 1, 1505 cm− 1, and 1650 cm− 1 in the PreS1 chart, respectively. Furthermore, the peaks at 1435 cm− 1 and 1061 cm− 1 imply a combination of C-O and C = O and a C-O-C stretching. Peaks 1606 and 1520 show (NH2 stretching), indicating that the amine groups in the CS NPs have bound to the phosphate [47,48]. Peak 1232 in CS NPs is related to the phosphate group (P = O and P-O) of TPP, which is transferred to 1264 after coating with PreS1. After coating, peaks 1564 and 1642 represent the first and second types of amide, which is a confirmation of the connection of PreS1 with the amino group of CS NPs (Nandiyanto et al. 2019; Roy et al. 2019; Taghipour-Sabzevar et al. 2020).

Fig. 6
figure 6

The viability rates of different drug delivery systems on HepG2 cells after 24 h (a) and after 48 h (b). A549 (Lung cancer cell) and Hu02 (Normal fibroblast) cells are as control (*P < 0.05 and **P < 0.001)

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Fig. 7
figure 7

Annexin-PI apoptosis diagram of HepG2 cells treated with PBS (a), Cs-NPs (b), Pres1-Cur-CM11-Cs NPs (3 µg/ml peptide concentration) (c), Pres1-Cur-CM11-Cs NPs (6 µg/ml) (d), Pres1-Cur-CM11-Cs NPs (12 µg/ml) (e), Pres1-Cur-CM11-Cs NPs (24 µg/ml) (f), and total apoptosis (g) (*P < 0.05-**P < 0.001)

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Cur and CM11 loading in NPs

Based on the results, drug loading content (LC) of Cur and CM11 in Pres1-Cur-CM11-CS NPs were 14% and 20%, while drug loading efficiency (LE) was 85% and 65%, respectively (Table 1). Drug loading content and drug loading efficiency are two important parameters of drug nanocarriers. The drug loading content reflects the ratio of the drug mass to the drug-containing nanocarrier, and the drug loading efficiency also indicates the amount of drug loading during the preparation process of the nanocarrier (Shen et al. 2017). In general, the drug loading content is determined by the structure and physical and chemical properties of the carrier material and drug loading efficiency is impacted by the drug loading mechanism, drug mass and other experimental conditions (Shen et al. 2017; Alshawwa et al. 2022). As is clear from the results of this study, it is more difficult to obtain high drug-loading content than high drug-loading efficiency for most nanocarrier systems. Studies showed that if the space capacity of the carrier is low, it is difficult to get high drug-loading content, even if the preparation process of the nanocarrier shows high drug-loading efficiency (Zhang et al. 2015; Ribeiro et al. 2017) which can be seen in this study so that for both drugs the amount of LC% is significantly less than LE%. However, high drug-loading efficiency may be necessary but not sufficient to achieve high drug-loading content (Shen et al. 2017). In most drug delivery systems, high amounts of carrier materials must be used to administer a proper dose of the drug, which may cause systemic toxicity. As such, FDA approval for most nanocarriers is challenging to obtain which has led to the limitation of their clinical applications. To solve this limitation, providing a multidrug codelivery system based on combined drugs can be beneficial. Given the advantages of combination therapy, including maximum therapeutic effect, minimum delay in induction of drug, and potential overcoming of multidrug resistance mechanisms of tumors, this method has become a new hot spot in the field of drug delivery systems (Azad et al. 2008; Blank et al. 2011). Accordingly, in this study, we used a multidrug codelivery system that carried two drugs including CM11 peptide and curcumin. As shown in Table 1 and in optimized conditions, LC% of the peptide and Cur in CS NPs (as single) is 22% and 30% and in PreS1-CS NPs (as dual) is 14% and 20%, respectively (Table 1), which is suitable for a drug release system. Generally, based on reports, for NPs with drug loading content higher than 50%, sustained-release effects are far below expected and with higher than 90% it is usually hard to achieve surface modification.

In vitro release study

During the treatment cycle, the primary goal of drug therapy for any disease is to obtain and sustain the drug’s optimal therapeutic concentration at the site of action. NPs can control the release of drugs over a long period, thus increasing the therapeutic index of the pharmacological activity of agents (Lee and Yeo 2015). Drug release from CS NPs is typically controlled by polymer swelling, drug diffusion, polymer erosion or degradation, and or a combination of both (Herdiana et al. 2022). In addition, CS degradation can be triggered by environmental conditions such as temperature, pH or enzymes. However, drug release from CS NPs is more pH-dependent (Mikušová and Mikuš 2021; Herdiana et al. 2022). Acidic pH increases the electrostatic attraction between the protonated amine groups of chitosan and H2O molecules, which leads to the degradation of the CS structure by increasing swelling and accelerating the release of the drug. The microenvironment of tumors is acidic (pH 5.6 to 6.8) due to glycolysis in tumor cells, hypoxia, and insufficient blood perfusion (Kulpreechanan and Sorasitthiyanukarn 2020; Herdiana et al. 2022). Glycolysis in cancer cells results in a high intracellular lactic acid concentration, therefore, cancer cells prevent this acid stress by activating and expressing proton and lactate transporters and exchangers. In this case, tumor cells have an inverse pH gradient, so that the extracellular and intracellular pH are acidic and alkaline, respectively. Based on this, in this study, drug release from CS NPs was evaluated at physiological (pH 7.4) and acidic pHs (pH 5.5) (de la Cruz-López et al. 2019; Bogdanov et al. 2022).

As shown in Fig. 5a, which is related to pH 7.4, free Cur and CM11 were completely released after 8 h, while at this time the release of Cur and CM11 from both coated and non-coated NPs was less than 55%, which indicates the controlled and sustained release of the drugs from NPs. At this time, the amount of Cur release from Cur-CS NPs and PreS1-Cur-CS NPs was 55% and 45%, respectively, but CM11 release was about 35%. On the other hand, the Cur and CM11 release from CS NPs and PreS1-CS NPs in the acidic pH (5.5) was higher compared to the physiological pH (p-value ≤ 0.05) (Fig. 5b). Accordingly, the amount of Cur release from non-coated and coated NPs was 70% and 65%, respectively, and for CM11 was 55% and 45%, respectively. After 48 h and at pH 7.4, the amount of release of Cur from CS NPs and PreS1-CS NPs was 60% and 80% while for CM11 was 40% and 55%, respectively, which indicates the slow and sustained release of drugs, which is also dependent on pH (p-value ≤ 0.05). It is necessary to explain that the amount of drug release per unit time from PreS1-CS NPs containing one drug (Cur or CM11) was not significantly different from PreS1-CS NPs containing two drugs (Cur + CM11) (p-value > 0.05). Furthermore, the results showed that the release of Cur is faster than the CM11 peptide, which is probably related to the size and molecular weight of these two drugs. The molecular weight of Cur and CM11 are about 368 and 1414 Da, respectively, which shows that the peptide is a larger molecule and therefore its release occurs in a slower process. Of course, the release rate of Cur and CM11 from CS NPs coated with PreS1 is almost less compared to non-coated CS NPs at the same time, which is probably due to the presence of PsrS1 (3133 Da) on the surface of NPs. This coating makes it harder for the drug to pass through and be released from the NPs. In addition, the net charge of the CM11 peptide is positive in physiological and acidic pH (+ 4, Iso-electric point at pH 11), while it is negative for PreS1 (-4, Iso-electric point at pH 3), and this can strengthen the electrostatic interactions between them and lead to a slower release of the CM11 peptide.

To simulate the Cur and CM11 release pattern from CS NPs, zero-order, first-order, Higuchi, and Korsmeyer-Peppas models were used and the DDsolver plugin was also used to verify the results (Zhang et al. 2010). The highest correlation coefficient (R2) for the release of Cur (0.94 for pH 7.4 and 0.93 for pH 5.5) and CM11 (0.95 for pH 7.4 and 0.97 for pH 5.5) from Pres1-Cur-CM11-CS NPs and the highest correlation coefficient (R2) for the release of Cur (0.92 for pH 7.4 and 0.92 for pH 5.5) and CM11 (0.96 for pH 7.4 and 0.90 for pH 5.5) from Cur-CM11-CS-NPs were related to the Korsmeyer-Peppas model (Formula 5).

$$\:\text{F}\hspace{0.17em}=\hspace{0.17em}\text{K}\text{k}\text{p}\:\times\:\:\text{t}\text{n}$$
(5)

Kkp = Rate constant; n = Diffusional exponent; t = Time; F = Release rate.

According to the values of n, which are around 0.2 to 0.4 in all models for Cur and CM11, with assumptions such as release in one dimension and the low length-to-thickness ratio of the designed systems, the drugs’ release can be considered under Fick’s law. According to Fick’s law of diffusion, drug molecules diffuse from a region of high drug concentration to a region of low drug concentration (Siepmann and Siepmann 2012).

Stability of NPs

The stability of a drug delivery system as one of the most important aspects of drug delivery needs to be measured before it can be used clinically because the drug loaded in the nanoparticles may be slowly released before reaching the tumor. It can increase the risk of side effects, immune system xenophagy, and other problems (Zhang et al. 2021). Based on this, drug-carrying nanoparticles must be stable in terms of drug release or aggregation until they reach the desired location. So, higher stability prolongs the duration of drug circulation and results in a more concentrated and targeted accumulation inside the tumor microenvironment (Guerrini et al. 2018). The stability of Pres1-Cur-CM11-CS NPs was investigated by measuring the particle size and surface charge of NPs. After 3 weeks, the particle size was checked using the DLS. After 21 days, the size of the NPs increased by about 15 nm and reached around 150 nm, which was not significant (p-value > 0.05) and can be said to be relatively stable (data not shown). As shown in the previous sections, the prepared NPs had a large positive surface charge, and there was a strong repulsive force between them that prevented the NPs from aggregating with each other. But after a few days, with the reduction of this surface charge, a slight increase in the size of nanoparticles occurs, which can be due to partial aggregation.

MTT assay

The cytotoxicity of Pres1-Cur-CM11-CS NPs, Pres1-Cur-CS NPs, Pres1-CM11-CS NPs, Cur-CM11-CS NPs, Cur-CS NPs, CM11-CS NPs, CS NPs, free Cur, and free CM11 were investigated on HepG2 cell lines after 24 h (Fig. 6a) and 48 h (Fig. 6b). In addition, A549 and Hu02 cell lines (Lung cancer cell) were used as control samples to evaluate the specificity and targeted release of drugs from Pres1-Cur-CM11-CS NPs. Concentrations were calculated based on the IC50 of CM11 peptide, and a range of concentrations of 3, 6, 12, and 24 µg/ml were used. Cur concentrations were also calculated based on its LC50 (based on reports) and relative to CM11 concentrations as 1.9, 3.8, 7.6, and 15.2 µg/ml, respectively. As shown in both Fig. 6a and b, the cell survival rate after 24 and 48 h for free CS-NPs with different concentrations is more than 93%, which indicates the appropriate biocompatibility of the CS-NPs. According to the findings, the anticancer activity of the peptide is more than that of curcumin, and for both drugs, this activity is dose-dependent in all groups. At higher concentrations of Cur (15.2 µg/ml) and CM11 (24 µg/ml) after 24 h, the Pres1-Cur-CM11-CS NPs showed the highest toxicity, with a survival rate of about 50% and had a significant difference (p-value ≤ 0.05) with more toxicity compared to the Cur-CM11-CS NPs with survival rate of about 60%, which shows that Pres1-Cur-CM11-CS NPs are more effective. Based on this, the survival rate of treated cells with PreS1-Cur-CM11-CS NPs after 24 and 48 h was 50 and 30%, respectively, while for Cur-CM11-CS NPs was 60 and 45%, respectively. After 24 and 48 h, the survival rate of treated cells with free drugs as dual was also 65 and 55%, respectively, which confirms that simultaneous encapsulation of two drugs in NPs can significantly improve their anticancer activity (p-value ≤ 0.05) and coating NPs with PreS1 peptide for targeted release also significantly increases this activity (p-value < 0.001). PreS1-Cur-CM11-CS NPs and Cur-CM11-CS NPs showed the most cytotoxic effects compared to other drug groups. Accordingly, the results demonstrated that nanoparticles carrying two drugs (Cur + CM11), whether coated with PreS1 (PreS1-Cur-CM11-CS NPs) or non-coated (Cur-CM11-CS NPs), have more anticancer activity than similar nanoparticles carrying a single drug (Cur or CM11). This proves that two drugs together have a synergistic effect, and on the other hand, encapsulation in non-coated NPs is also significantly effective in increasing their anticancer activity (p-value ≤ 0.05), because as previously presented, non-coated nanoparticles have a significant positive charge that allows them to effectively interact with the membrane of cancer cells that have a negative charge. Of course, targeting nanoparticles using PreS1 peptide leads to a more effective release of these two drugs, which can be due to the uptake of PreS1-coated CS NPs by receptor-mediated endocytosis depending on the interaction between PreS1 and SB3 antigen. For A549 and Hu02 cells as control, it has been shown that although the cytotoxic effect of drugs encapsulated in nanoparticles is greater than in the free form, no significant difference was observed between coated (PreS1-Cur-CM11-CS NPs) and non-coated (Cur-CM11-CS NPs) delivery system. This proves that for these cells, due to the absence of SB3 antigen, NP coating has no effect on increasing their effectiveness. However, the viability of Hu02 cells was higher than A549 cells, which is probably due to the acidification of the culture medium of A549 cancer cells, which leads to more drug release at the same time. In addition, the survival rate of these cells in the face of coated nanoparticles was higher than the case when uncoated nanoparticles were used, which is probably because the surface coating of nanoparticles causes the drug to be released in a slower process. It should be noted that the synergistic effect of these two drugs is significant because their cytotoxic effect when they are used freely and together is not significantly different from the case where each drug is encapsulated in nanoparticles (coated and non-coated CS NPs) alone.

The results of this study were similar to another report by Taghipour-Sabzevar, et al., which CM11 peptide was targeted to CD44 overexpressing A549, SH-SY5Y, and PANC-1 tumor cells using hyaluronic acid-coated chitosan nanoparticles (HA-CS NPs) (Taghipour-Sabzevar et al. 2020). Hyaluronic acid has a tumor-targeting property that specifically tends to bind to the CD44 antigen as a membrane glycoprotein overexpressed on the surface of many cancer cells (Taghipour-Sabzevar et al. 2020; Akim et al. 2021). The particle size and zeta potential of HA-CS NPs were 190 nm and + 28.9 mv with a reasonable PDI of 0.280. In addition, the encapsulation efficiency of peptide in CS NPs was estimated at approximately 60%. These results are similar to the current study, however, the size of HA-CS NPs is larger (190 nm) compared to the PreS1-CS NPs (132 nm), which is probably due to the structure of hyaluronic as a macromolecule in comparison with PreS1 peptide. The in vitro release of the CM11 peptide from HA-CS NPs was also pH-dependent, which was higher in acidic pH. Based on the MTT assay, the cytotoxicity of HA-CM11-CS NPs against cancer cell lines was significantly higher compared to the CM11-CS NPs and free peptide, which is similar to the results in this study. Also, in a similar study with a passive targeting approach by Prasad et al. in vitro anticancer activity of curcumin-loaded chitosan nanoparticles (CLCNPs) against Vero cells was investigated at different concentrations of Cur (10, 20, and 50 µg) (Prasad et al. 2022). NPs have a size ranging from 114 to 160 nm and it was observed that 50 µg concentration was most effective with the decreasing viability to 76%, while at 10 and 20 µg concentrations of CLCNPs, the percent viability was 92% and 85%, respectively. In a study by Huang et al. they developed curcumin-loaded galactosylated chitosan-coated nanoparticles for targeted delivery of hepatocellular carcinoma (Huang et al. 2023). This nanocarrier was a galactosylated chitosan (GC)-modified nanoparticles (GC@NPs) based on poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) (PEG-PLGA), which can target asialoglycoprotein receptor (ASGPR) expressed on HCC cells. For the fabrication of Cur-loaded NPs different concentrations of the Cur was used from 7.5 to 30 µg/ml. Their results showed that the drug loading (DL) of Cur was about 4.56%. Furthermore, in vitro cellular assays confirmed that GC@NPs could be efficiently internalized to HepG2 cells via ASGPR-mediated endocytosis, leading to superior inhibition of tumor growth compared to free Cur. Similar to our findings, the results proved that with the increase of incubation time and total drug concentration, the cytotoxicity of all drug groups (Free Cur, CS NPs, and GC NPs) increased. The higher inhibition rate on HepG2 cells was for GC@NPs which may be due to the fact that GC@NPs introduce GC groups that are specifically recognized by HepG2 cell surface receptors and thus are selectively taken up by HepG2 cells. Also, based on the anticancer activity of antimicrobial peptides and targeting them against HCC, Wu et al. investigated a cell transmembrane peptide chimeric M(27–39)-HTPP targeted therapy for HCC (Wu et al. 2023). Heparin sulfate proteoglycan (HSPG) is a macromolecule found mainly in the cell surface and extracellular matrix of liver tissue cells (Roskams et al. 1995). Glypican3 (GPC3) is a member of this family, which is reported to be abundant in HCC tissue, but absent in normal liver tissues (Zhou et al. 2018). HTPP is a liver-targeted cell-penetrating peptide (CPP) derived from the circumsporozoite protein (CSP) of Plasmodium parasites. Studies have shown that HTPP peptide can specifically bind to GPC3 and penetrate the cell membrane of liver cells (Wu et al. 2023). So. HTPP can be a suitable tool for specific targeting HCC cells. Similar to the CM11 peptide as an antimicrobial peptide, the M27-39 peptide is an antimicrobial peptide derived from the antimicrobial peptide Musca domestica cecropin (MDC) which is obtained from M. domestica larvae. The M27-39 peptide has higher anti-HCC activity and smaller molecules (Wu et al. 2023). Accordingly, by fusion of HTPP and M27-39, they produced a new peptide based on the targeted penetration of HTPP and the anti-tumor effect of M27-39 against HCC cells. Analyses revealed that M(27–39)-HTPP had a good ability to target and penetrate the HCC cells, effectively limit the proliferation, migration, and invasion, and induce apoptosis in HCC in vitro and in vivo. Compared to the current study, after 24 and 48 h of treatment by hybrid peptide at 320 µg/ml concentration, cell viability was about 66% and 37%, respectively, while for PreS1-Cur-CM11-CS NPs was 50% and 30%, respectively. Based on the cell survival rate, it can be concluded that our drug delivery system prevents the growth of HCC cells more effectively. Also, the dose of the peptide drug used is lower compared to the study by Wu et al. On the other hand, drug delivery based on nanoparticles leads to greater drug stability in the face of environmental factors. Finally, it should be mentioned that in the basic study of the possibility of targeting the drug against HCC cells using the specific interaction of PreS1 peptide and SB3 antigen, Jha et al. evaluated 2 nm (metal core) gold nanoparticles to target HCC (HepG2) cells which were coated with a 85:15 mixture of thiols featuring, respectively, a phosphorylcholine (to ensure water solubility and biocompatibility) and PreS1 peptide (PreS1-Au NP). Their results confirmed that the PreS1 peptide is a suitable targeting agent for HCC cells overexpressing the SB3 protein. Analyses showed that the binding of PreS1-Au NPs is more than 6 times higher than that of the nanoparticles without the peptide (Au NPs) (Jha et al. 2017).

Apoptosis assay

Annexin-PI staining was used to investigate apoptotic or neurotic effects of PreS1-Cur-CM11-CS NPs at different concentrations of CM11 peptide (3, 6, 12, and 24 µg/ml) on HepG2 cells (Fig. 7a-g). The results showed that after 24 h the viability rate of cells treated with CS-NPs was 90%, and the biocompatibility of the delivery system was confirmed. In Fig. 7a-f, the Q2 section represents late apoptosis, and Q3 represents early apoptosis. Accordingly, total cell apoptosis is the sum of Q2 and Q3. Based on analyses, cell apoptosis increased significantly (p-value ≤ 0.05) with the increase in drug concentration in a dose-dependent manner and was equal to 14.42, 27.65, 34.8, and 38.78%, respectively, which proves that PreS1-drugs-CS NPs leads to the induction of apoptosis in cells., while at different concentration cell necrosis was around 1 to 2%.

Conclusion

Targeted drug delivery has significant advantages over conventional drug delivery methods. This method leads to a targeted increase in the concentration of the drug at the desired site, such as organs, tissues or cells, while reducing the release of the drug in other areas of the body. This targeted approach improves treatment efficacy while minimizing side effects. Additionally, systems based on targeted drug release can help improve therapeutic outcomes by prolonging drug release, localizing its effect, and protecting it during delivery. In addition, this method can even lead to the development of new drug classes. Targeted delivery systems can expand the administration of drugs that were not previously possible to use safely and effectively, thus potentially enabling the development of new treatments for previously untreatable conditions. The side effects of chemotherapy drugs and the possibility of drug resistance are among the risks of cancer chemotherapy. Reducing the dosage can be effective in reducing this risk. As mentioned, targeted release can be a suitable method to reduce the drug dosage and increase its therapeutic potential. In this study, we showed that the use of PreS1 peptide-coated chitosan nanoparticles can significantly increase the anticancer effect of CM11 peptide and curcumin against HCC cells. On the other hand, the simultaneous use of these drugs also increases their anticancer properties. In general, this study confirms that by using the PreS1 peptide, the drug release system can be targeted specifically against HCC cells, and the use of the CM11 peptide should be considered as an alternative anticancer drug. However, more studies are needed in vivo.