Dihydroxyphenylalanine-conjugated high molecular weight polyethylenimine for targeted delivery of Plasmid

Abstract

High molecular weight polyethylenimine (HMW PEI; branched 25 kDa PEI) has been widely investigated for gene delivery due to its high transfection efficiency. However, the toxicity and lack of targeting to specific cells have limited its clinical application. In the present investigation, L-3, 4‐dihydroxyphenylalanine (L-DOPA) was conjugated on HMW PEI in order to target L-type amino acid transporter 1 (LAT-1) and modulate positive charge density on the surface of polymer/plasmid complexes (polyplexes). The results of biophysical characterization revealed that the PEI conjugates are able to form nanoparticles ≤ 180 nm with the zeta potential ranging from + 9.5–12.4 mV. These polyplexes could condense plasmid DNA and protect it against nuclease digestion at the carrier to plasmid ratios higher than 4. L-DOPA conjugated PEI derivatives were complexed with a plasmid encoding human interleukin-12 (hIL-12). Targeted polyplexes showed up to 2.5 fold higher transfection efficiency in 4T1 murine mammary cancer cell line, which expresses LAT-1, than 25 kDa PEI polyplexes prepared in the same manner. The cytotoxicity of these polyplexes was also substantially lower than the unmodified parent HMW PEI. These results support the use of L-3, 4‐dihydroxyphenylalanine derivatives of PEI in any attempt to develop a LAT-1 targeted gene carrier.

Introduction

In recent decades, great attention has been directed to gene and drug delivery using nanotechnology-based materials^1–4. The approval of covid-19 mRNA vaccines as well as some siRNA-based medications using lipid nanoparticles has advanced the bench to bed translation of nano-delivery systems for nucleic acid materials^5–8.Although viral delivery of nucleic acids has demonstrated great achievements, there are several concerns limiting their wide use. These limitations including limited gene carrying capacity, stimulation of the host immune system, the possibility of insertional mutations and high production costs have led researchers to seek alternative approaches for gene delivery⁹. Among various non-viral gene delivery strategies, polycationic polymers have attracted substantial attention. These gene delivery vehicles can be tailored through the optimization of their biophysical and biological characteristics by various methods including conjugation of the targeting ligands. Functionalization of polymeric gene carriers by targeting ligands leads to the accumulation of targeted vehicles in the desired site of action and minimizes the side effects associated with their wide distribution to the other organs or tissues^10–14.

Polyethylenimine (PEI) is one of the widely-investigated polycationic compounds for nucleic acid delivery¹⁵. The properties of this polymer enabling it for gene delivery include high cationic charge density and the ability to induce early escape from endosomal compartments before enzymatic degradation. PEI condenses nucleic acid materials into nano-sized particles, namely polyplexes and protects the payloads from nuclease degradation. However, the high positive charge and non-targeted distribution of PEI has limited its clinical application despite its high transfection efficiency¹⁶. Therefore, the modulation of cationic charge as well as conjugation of targeting ligands on its structure have been considered as promising approaches to overcome the major barriers towards its wider in vivo applications¹⁷.

Various targeting ligands have been conjugated on PEI including small molecules, peptides, aptamers as well as monoclonal antibodies^18,19. L-type amino acid transporter 1 (LAT-1) is one of the transporters to deliver large neutral amino acids (e.g.; isoleucine, methionine, tryptophan, phenylalanine, leucine, cysteine, tyrosine and glutamine) as well as drugs across the biological barriers such as blood–brain barrier (BBB), placenta, testis and various cancer tissues²⁰. The two subunits of LAT-1 are covalently connected via a disulfide bond. The exchange of amino acids is carried out by the light chain of the transporter while the heavy chain acts as a chaperone and localizes the light chain in the plasma membrane²¹. Since this transporter has shown the ability for delivery of some drugs such as L-DOPA (L-3,4-dihydroxyphenylalanine), melphalan and gabapentin to their site of action, the conjugation of these molecules on gene delivery vehicles may direct them to the cells or tissues expressing LAT-1 and enhance the therapeutic efficacy in the desired site of action. The conjugation of different nanoparticulate systems with LAT-1 ligands have been carried out in some studies. For instance, liposomes, gold nanoparticles and polymeric carriers have been decorated with glutamate and L-DOPA^22,23.

L-DOPA is an amino acid molecule, which is naturally made from the biosynthesis of L-tyrosine in the human body. This molecule has shown the ability to cross the BBB and cancer cells’ membrane through LAT-1. There are several reports indicating the role of L-DOPA conjugation on nanoparticles to increase their delivery into cancer cells. For instance, functionalization of polyoligoethylenimine (pOEI) with L-DOPA enhanced the accumulation of targeted nanoparticles in the human-derived malignant glioma cells U87²⁴. In another investigation, the injection of L-DOPA containing liposomal formulations resulted in the accumulation of the carrier in the brain confirming the role of LAT-1 in the transport of the vehicle across BBB²⁵.

In recent years, immunotherapy has been proposed as a new paradigm for cancer treatment. Therefore, great attention has been directed to cytokines with potential anticancer effects^26,27. Interleukin-12 (IL-12) has shown promising antitumor properties in animal studies as well as robust immunostimulatory effects in human clinical trials²⁸. However, its clinical application in humans has been limited due to the severe systemic reactions following administration of the recombinant human IL-12 protein²⁹. As an alternative strategy, it has been suggested to transfer the plasmid encoding IL-12 gene into the precise site of action^30,31. In this method, expression of the desired therapeutic protein occurs in the target organ that may limit the adverse reactions associated with the systemic administration of the protein. This therapeutic approach can particularly be used for local tumors such as melanoma. For instance, an intratumoral formulation of hIL-12 mRNA was successfully applied for melanoma treatment^32,33. These findings highlight the potential future application of nucleic acid-based approaches for hIL-12 therapy³⁴.

In the present study, L-DOPA was conjugated on PEI and the polyplexes were prepared by interaction between the modified PEI conjugates and plasmid encoding hIL-12. The polyplexes were characterized in terms of particle size, zeta potential, plasmid condensation ability as well as protection against nuclease digestion. Finally, the transfection efficiency and cytotoxicity of the polyplexes were evaluated on the cells expressing LAT-1 and compared with the control cells.

Materials and methods

Materials

Branched polyethylenimine (PEI; average MW 25 kDa), L-3,4-dihydroxyphenylalanine (L-DOPA), N-Hydroxysuccinimide (NHS), N,N-Dicyclohexylcarbodiimide (DCC) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (Munich, Germany). Human IL-12 plasmid (pUMVC3-hIL-12) was obtained from Aldevron (Madison WI). Human IL-12 ELISA kit was purchased from BD Bioscience (Heidelberg, Germany). Fetal Bovine Serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Gibco (Gaithersburg, MD, USA). KBC Power Load was purchased from Kawsar Biotech Company (Tehran, Iran). DNA ladder 1 kb and DNase I were purchased from Cinnagen 8 (Tehran, Iran). Dialysis was carried out using Spectra/Por dialysis membranes (Spectrum Laboratories, Houston, TX USA). All solvents and chemicals were purchased from Sigma-Aldrich (Munich, Germany) and were of the highest purity available.

Synthesis of L-DOPA-PEI conjugates

In order to synthesize L-DOPA-PEI conjugates, desired amounts of L-DOPA was added to dimethyl sulfoxide (DMSO) and allowed to be dissolved for 30 min with shaking. In order to activate the carboxylic acid group of L-DOPA, 1 mol equivalent of N,N-Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were added to the solution with constant shaking and the reactions were carried out for 24 h at room temperature. In order to achieve 1, 2 and 4% substitution of PEI primary amines, 25 kDa branched PEI (150 mg) was dissolved in 30 mL DMSO. The desired concentration of L-DOPA solution necessary for the preparation of PEI derivatives with calculated conjugation degrees (i.e.; 1, 2 and 4% of primary amines) was added dropwise to the PEI solution with constant shaking and the reaction was carried out for 24 h at room temperature. Finally, the crude product was dialyzed (1000 Da cut-off Spectra/Por membrane) against distilled water three times. After dialysis, the solutions were lyophilized and the fluffy powders were characterized by FT-IR spectrometry using Bruker IR spectrometer (Germany) and ¹H-NMR (D₂O) spectrometry using a Bruker Avance DRX-500 MHz NMR spectrometer (Bruker, Ettlingen, Germany)³⁵. The modified PEI derivatives were labeled as PEI–LD% in which LD represents L-DOPA and % is the percentage of PEI amines grafted by L-DOPA.

Preparation of plasmid

The plasmid (pUMVC3-hIL12) was transformed into Escherichia coli bacterial strain DH5α, incubated in Luria–Bertani (LB) media, then extracted and purified from the culture pellets using the FavorPrep Plasmid Extraction Maxi Kit (Favorgen, Taiwan) according to the manufacturer’s instruction. The purity and concentration of the plasmid were assessed using a UV–Vis spectrophotometer at 260, 280 and 320 nm. In addition, the size and integrity of the plasmids were confirmed using agarose gel electrophoresis.

Preparation of polyplexes

In all experiments, the composition of polyplexes was characterized by C/P ratio in which C is the weight of PEI and its conjugates and P represents the weight of the plasmid DNA used for the complex formation. PEI and conjugates were prepared at different concentrations and added to the same volume of plasmid DNA solution (40 μg/ml) in separate tubes in HBG buffer (HEPES buffered glucose solution; 20 mM HEPES, 5% glucose, pH = 7.4). Then the solutions were mixed and incubated for 10 min at room temperature to form stable complexes.

Evaluation of buffering capacity

Buffering capacity is known as the number of moles of acid or base that must be added to change the pH by one unit in 1 L of solution. A simple acid–base titration method was used to measure the buffering capacity of PEI and its derivatives³⁶. Solutions of PEI and conjugates (0.4 mg/mL) were prepared in water. pH was adjusted to 12 using NaOH (1N). Then, 3 μL of HCl (1N) was added and pH of the solution was measured after each addition until the pH value was reached to around 2.5.

Measurement of particle size and zeta potential

To measure size and zeta potential of the polyplexes, desired amounts of PEI and its conjugates in HBG buffer were added to the plasmid DNA solution prepared in the same buffer. The size and zeta potential of the polyplexes were measured by Dynamic Light Scattering (DLS) and Laser Doppler Velocimetry (LDV), respectively, using nanoparticles size analyzer, Horiba Sz-100 (Japan). The measurements were carried out in automatic mode and the results were presented as mean ± SD, n = 3.

Plasmid DNA condensation assay

The ability of PEI and conjugates to condense pDNA was studied by gel electrophoresis. The polyplexes were prepared at various C/P ratios ranging from 0.5:1- 8:1. Then, 10 μL of each polyplex was loaded onto 1% agarose gel and electrophoresis was carried to visualize plasmid DNA bands³⁷.

Nuclease protection assay

To assess the capability of PEI and its derivatives to protect plasmid DNA against enzymatic degradation, DNase I protection assay was carried out as described in the literature. The polyplex formulations were prepared as described earlier and treated with 1 μL of DNase I enzyme. The same polyplex formulations were treated with PBS as negative control. The plasmid bands were visualized following agarose gel electrophoresis at 100 V for 1 h ³⁸.

Cell culture and cell viability assay

Human HepG2 hepatocellular carcinoma cells (NCBI C158, Tehran, Iran) and 4T1 murine mammary tumor cell line (NCBI C135, Tehran, Iran) were purchased from National Cell Bank of Iran (NCBI) and maintained at 37 °C, 5% CO₂ in DMEM supplemented with 10% FBS, streptomycin at 100 μg/mL, and penicillin at 100 U/mL. One day before starting toxicity studies, cells were seeded at the density of 1 × 10⁴ cells/well in 96-well trays for 24 h. The cytotoxicity of serial dilutions of PEI and its derivatives at concentrations ranging from 2 to 100 μg/mL was measured using MTT assay. Ten microliters of each concentration was added to the wells and incubated at 37 °C for 4 h. Then, the medium was replaced with 100 μL fresh medium. Forty-eight hours later, the medium was aspirated and the MTT solution (5 μg/mL) was added to each well and incubated for 2 h. The formazan crystals were dissolved in 100 μL/well dimethyl sulfoxide (DMSO) and the absorbance was measured by an ELISA reader at 590 nm and background was corrected at 630 nm. Data were presented as mean ± SD; n = 3.

In vitro hIL-12 delivery experiments

To evaluate the ability of polymers in transferring the plasmid encoding human IL-12 gene, the polyplexes were prepared at C/P ratios of 0.5:1, 4:1, and 8:1. Then, 10 μL of each polyplex formulation (equivalent of 200 ng plasmid DNA) was added to the wells and incubated at the same conditions as described for cytotoxicity assay. After 48 h, hIL-12 quantification was carried out by collecting the cell supernatants. The level of hIL-12 in the cell supernatants was measured by human IL-12 (p70) ELISA kit (BD Bioscience, Heidelberg, Germany) according to the manufacturer’s protocol. The results of hIL-12 expression were normalized and presented as pg/mL/seeded cells as described by Lotfipour et al³⁹. Three controls including polymer itself, plasmid DNA (without polymer) and cells without any plasmid DNA, polymer or polyplex were used to show the background value. All transfection experiments were carried out under FBS condition.

Transfection specificity test

In order to demonstrate the impact of L-DOPA conjugation in targeted delivery of PEI derivatives, inhibition of transfection was carried out using free L-DOPA. 4T1 cells were seeded as described above and pretreated with L-DOPA at concentration of 10^–3 mol/mL of the free ligand for 30 min at 37 °C. Then, the polyplexes were added into the medium and incubated for 4 h. Following the medium replacement, the incubation continued and hIL-12 quantification was carried out 48 h later³¹.

Statistical analysis

All data were presented as mean ± SD. The statistical significance was determined using Student’s t-test, and p values ≤ 0.05 were considered significant.

Results and discussion

Synthesis of L-DOPA-PEI conjugates

In order to prepare PEI derivatives, L-DOPA was conjugated to PEI structure using amide bond formation as described elsewhere. Following the conjugation, the structure of PEI conjugates was characterized by FT-IR and ¹H-NMR spectroscopy. According to FT-IR spectra (data not shown), the amide peak appeared at 1642–1653 cm⁻¹. This peak shows the formation of amide linkage between the PEI amine and L-DOPA carboxylic acid groups (Fig. 1). ¹H-NMR spectra demonstrated the peaks at 7.7–7.9 ppm that was assigned to the aromatic protons of L-DOPA, while the PEI protons appeared between 2.2 and 3.2 ppm. The modification degree was analyzed by ¹H-NMR in D₂O on a Bruker Avance DRX-500 MHz NMR spectrometer from the ratio between the peaks of PEI (δ 2.2–3.2 ppm) and aromatic protons of L-DOPA (δ 7.7–7.9 ppm). The modification degree was expressed as the number of modification per monomer unit of PEI × 100%.The conjugation degree was calculated by ¹H-NMR and resulted in the formation of three conjugates namely, PEI-LD 1%, PEI-LD 2% and PEI-LD 4% (FT-IR and ¹H-NMR spectra are available in Supplementary Fig. S1–S8 online).

Fig. 1.

Synthesis of L-DOPA grafted PEI derivatives. Branched PEI (25 kDa) was modified by Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS). PEI and L-DOPA were conjugated via an amide bond.

Evaluation of buffering capacity

In order to measure the buffering capacity of PEI and its conjugates, an acid–base titration method was carried out. The intrinsic capacity of the PEI and conjugates to act as a buffering agent was shown by plotting the changes of pH versus the added amounts of acid. As demonstrated in Fig. 2, unmodified PEI demonstrated remarkable buffering capacity over almost the entire pH range. As indicated in Fig. 2, the buffering capacity was reduced by increasing the conjugation degree. In other words, buffering capacity was reduced with increasing the substitution degree.

Fig. 2.

Buffering capacity measurement. Titration curves for water, unmodified PEI and its derivatives from pH 12 to around 2.5.

Following cell internalization, polyplexes may enter endo/lysosomal compartments due to their route of cell entrance^40,41. Since these vesicles contain acid-hydrolases with the ability to degrade nucleic acid materials, they can act as a major barrier for efficient gene transfer. According to the proton sponge hypothesis⁴², PEI is able to act as a sponge to absorb protons and act as a buffering agent in the endosomal compartments. The capture of protons during the acidification of endosomes leads to the rise of osmotic pressure and disruption of the vesicle membrane. The result of buffering capacity measurement (Fig. 2) demonstrated a shift to the left following the conjugation of PEI with L-DOPA. Since PEI amines are in charge of the induction of the proton sponge effect, their utilization for conjugation decreases the intrinsic buffering capacity of the polymer. However, the critical buffering capacity for the induction of early escape from the vesicles is around 5–7 which acts as a driving force for the release of payload from endosomes to the cytosol⁴³. There are several studies indicating the reduction of buffering capacity following the conjugation of moieties via the amines of PEI. This may reduce the overall capacity of the modified polymer to induce early escape. Therefore, it is crucial not to waste the amines of PEI as conjugation points for ligands^44,45. It is suggested that preserving the conjugation degree at the lowest degrees may preserve the buffering capacity while modify the PEI characteristics such as targeting ability.

Particle size and zeta potential measurement of polyplexes

Polycationic polymers are able to interact with the negatively charged phosphate groups in the backbone of nucleic acid to form nano-sized particles. The formation of these so-called nano complexes is a crucial step for gene delivery⁴⁶. In this investigation, the size of modified PEI complexes were measured at C/P = 8 in HBG buffer. The results of particle size measurement (Table 1) showed that all conjugates are able to form nanoparticles smaller than 200 nm. The size range of 20–200 nm plays a significant role in the internalization pathway of polyplexes. In other words, maintaining the size in the ranges lower than 200 nm is a promising way to control the internalization of nucleic acids by polycations. Although the smallest size (i.e. 45 nm) was formed by unmodified PEI, the conjugated PEI derivatives formed larger polyplexes in which the size was increased by increasing the conjugation degree. The largest polyplex (PEI-LD 4%) formed the polyplexes with the size of 180 nm while the PEI-LD-1% resulted in the formation of polyplexes with the size of 80 nm⁴⁷. Regarding the zeta potential of polyplexes, unmodified PEI led to the formation of polyplexes with zeta potential of 16.3 ± 0.25 mV whereas the highest conjugation degree (PEI-LD 4%) led to the lowest zeta potential (i.e. 9.5 ± 0.25 mV) (Table 1). In other words, increasing the conjugation degree leads to the decrease in zeta potential. Overall, the remaining positive charge on the polyplexes is sufficient to induce adsorptive endocytosis. The reduction of positive charge following the conjugation of ligands through PEI amines has been reported in several studies^48–50. The most considerable point is to preserve the positive charge density of the polyplexes while modulating this positive charge may lead to lower toxicity (vide infra). The positive charge of polycationic carriers is essential for the formation of polyplexes and subsequently protection of nucleic acid materials as well as cell entry while the high charge density may lead to induction of toxic effects. Hence, modulation of charge density by conjugation of various ligands or moieties is a promising approach, which has shown substantial effect in the generation of polyplexes with lower toxic effects. On the other hand, the remaining positive charge on the conjugates is crucial to prevent particle aggregation leading to colloidal stability. In the previous investigations, it has been shown that conjugation of small molecule targeting ligands at the conjugation degrees around 1–10% of PEI amines results in the formation of polyplexes which have sufficient positive charge preventing their aggregation^51–53.

Table 1.

Size and zeta potential of the polymer/plasmid DNA complexes. Polyplexes were formed at C/P ratio of 8 in HBG buffer. Size and zeta potential measured by Dynamic Light Scattering (DLS) and Laser Doppler Velocimetry (LDV), respectively.

Materials	Size			Zeta potential (mV) Mean ± SD
	Intensity (nm) Mean ± SD	Volume (nm) Mean ± SD	Number (nm) Mean ± SD
PEI 25 kDa	48.8 ± 0.33	59.3 ± 0.37	51.7 ± 0.41	16.3 ± 0.25
PEI-LD 1%	77.5 ± 0	131.3 ± 0.05	119 ± 1.06	12.4 ± 0.55
PEI-LD 2%	83.5 ± 5.89	257.06 ± 1.3	129.1 ± 0.7	11.3 ± 0.11
PEI-LD 4%	177.9 ± 0.98	464.03 ± 3.72	339.5 ± 1.15	9.5 ± 0.25

DNA condensation assay

To investigate the ability of PEI and its conjugates to condense plasmid DNA into nano-sized particles, DNA condensation assay was conducted. As demonstrated in Fig. 3 and Supplementary Figs. S9 and S10 online, unmodified PEI could completely condense plasmid DNA at the lowest carrier to plasmid ratio (C/P = 0.5) while the conjugates were not able to condense pDNA at the same C/P ratio. At C/P ratios of 4 and 8 either unmodified PEI or PEI derivatives could completely condense plasmid DNA. By increasing C/P ratio, positive charge is provided resulting in the full plasmid DNA condensation. The condensation of nucleic acids to nano-sized particles is essential for gene delivery. However, there are controversial reports on the effect of polyplex tightness on efficiency of gene delivery^54–56. Although the interaction of polymeric compounds with nucleic acids and “DNA association” is a prerequisite step for successful DNA delivery, these complexes must be able to release their cargo (e.g. plasmid DNA) inside the cells. In other words, “dissociation” of plasmid DNA from their carrier leads to their transcription and finally protein production inside the cells⁴⁰. In the present study, the transfection efficiency of PEI derivatives at the lowest C/P ratio, which led to incomplete condensation, was higher than unmodified PEI at the same C/P ratio. The results of plasmid condensation was similar in the range of C/P = 0.5–4 as it was observed in the previous investigations^30,31,43,57. This result shows that incomplete condensation does not necessarily leads to lower transfection efficiency. This behavior might be justified by the fact that looser polyplexes and easy dissociation may also lead to efficient vector unpackaging and higher transfection efficiency compared with unmodified PEI at the same C/P ratio⁵⁸.

Fig. 3.

DNA condensation assay by agarose gel electrophoresis at various C/P ratios. The polymer and plasmid complexes were prepared at various C/P ratios ranging from 0.5:1 to 8:1. Then, each of them were incubated by KBC power load, loaded onto 1% agarose gel and electrophoresis was carried to visualize plasmid DNA bands. Full-length images as they are, with membrane edges visible are available in Supplementary materials (Supplementary Figs. S9 and S10).

Nuclease protection assay

In order to assess the ability of PEI and conjugates to protect plasmid DNA against enzymatic degradation, DNase I was used as the model enzyme. Protection effect of the polymers on plasmid DNA was demonstrated by agarose gel electrophoresis. One of the most important barriers for nucleic acid administration is stability in biological media. The presence of various nucleases in extracellular matrix and cytosol leads to the degradation of nucleic acid materials. Hence, protection of these materials via their complexation by electrostatic interaction with polycations is an effective strategy for in vivo applications. Polyplexes were prepared at C/P ratios of 0.5, 4 and 8 and treated with DNase I while the same polyplexes treated with PBS buffer were considered as control. As illustrated in Fig. 4 and Supplementary Figs. S11 and S12 online, neither unmodified PEI nor conjugates could protect plasmid DNA at C/P = 0.5 whereas significant protection effect at higher C/P ratios (e.g. C/P = 4 and 8) was observed. In our study, there is a consistency between the results of plasmid condensation and protection. In other words, the full condensation led to the complete protection against enzymatic degradation while the incomplete condensation results in insufficient protection. However, there are some controversial investigations reporting that complete condensation of pDNA does not lead to complete protection against enzymatic degradation due to the orientation of nucleic acids in the spaghetti-meatball-like structure of polyplexes^59,60.

Fig. 4.

DNase I protection assay by agarose gel electrophoresis at various C/P ratios. PBS (negative control) and DNase I (positive control) treated polyplexes in various C/P ratios. Then, they treated by EDTA and SDS, loaded onto 1% agarose gel and electrophoresis was carried to visualize plasmid DNA bands. + and − symbols represent the presence and the absence of DNase I enzyme; respectively. Full-length images as they are, with membrane edges visible are available in Supplementary materials (Supplementary Figs. S11 and S12).

Cell viability study

Cytotoxicity of the conjugates was evaluated by MTT assay on HepG2 and 4T1 cell lines (Fig. 5). The results of cytotoxicity on HepG2 cells revealed that PEI conjugates induce significantly lower toxicity in these cells. For example, the cell viability is around 80% even at the highest concentration of 100 μg/mL while the viability for unmodified PEI at the same concentration is around 10%. The substantial increase in the viability of cells might be the result of charge modulation on the surface of PEI polyplexes following conjugation.

Fig. 5.

Viability studies of conjugates by MTT assay. (a) and a Demonstrate the viability of conjugates at each concentration compared to PEI at the same concentration on HepG2 and 4T1 cell lines, respectively. ^ap-value < 0.05, ^bp-value < 0.001 & ^cp-value < 0.0001. (N = 3; error bars represent ± SD). (c) Indicates cell viability results of each conjugate compared to itself at the same C/P ratio on HepG2 and 4T1 cell lines, simultaneously.

According to the results (Fig. 5a), the toxicity of unmodified PEI occurred in a concentration-dependent manner in which the highest toxic effects were observed at the highest tested concentration. In other words, unmodified PEI at the concentration of 100 μg/mL induced about 90% toxicity in HepG2 cells while the lowest concentration of the same polymer (i.e. 2 μg/mL) resulted in no toxicity in the same cell line. The same results were also observed in 4T1 cells (Fig. 5b). The overall results of the viability study (Fig. 5c) revealed that toxicity of PEI conjugates is significantly lower than the unmodified parent polymer. This observation is consistent with several previous investigations in which the conjugation of various moieties via PEI amines reduced the cell-induced toxicity of the polycationic polymer^61–63. Since the major mechanism for the induction of toxicity by polycations is associated with the high amine charge density of the polycations, conjugation of different moieties through these amines decreases the toxicity. Reduced toxicity is mediated via reducing the probability of cellular damages resulting from the interaction of these positive charges not only with plasma membrane but also with the cellular compartments such as mitochondria^64,65.

The second probable reason for the decreased toxicity of PEI conjugates compared with the unmodified PEI is the mechanism by which the polyplexes enter the cells. There are several studies showing that the conjugation of targeting ligands reduces the toxicity of polyplexes by promoting the cell entry through a receptor or transporter mediated pathway. Since the targeted polyplexes enter the cells via a specific targeted mechanism, they remain in the cell culture medium shorter than non-targeted polyplexes. Therefore, they may have lower chance for disturbing interactions with the negatively-charged components on the cell membrane resulting in the cell damage and death¹⁶.

Gene transfer experiment

In order to evaluate the ability of PEI and its conjugates in transfection of plasmid encoding hIL-12 gene, the level of expressed hIL-12 was measured using an ELISA kit in 4T1 cells over-expressing LAT-1 transporter and HepG2 cell line lacking the transporter. As shown in Fig. 6, the conjugates resulted in the higher levels of hIL-12 production compared with the unmodified parent PEI. As illustrated in Fig. 6a, the level of hIL-12 production in HepG2 cells was not changed using unmodified PEI at different C/P ratios ranging from 0.5 to 8. However, the level of the protein was increased by PEI-LD 2% and 4% while it was not enhanced by PEI-LD 1%. Since there is no specific transporter for these conjugates on HepG2 cells, the reason for increased transfection efficiency could not be associated with transporter-mediated delivery. It seems that the conjugation of LD moieties on the surface of PEI reduces the positive charge density of the polyplexes (Table 1) leading to lower levels of cell-induced toxicity (Fig. 5). Since a higher number of live cells are present in the media, the level of protein production increased and higher transfection efficiency was observed.

Fig. 6.

The level of hIL-12 on HepG2 and 4T1 cell line following the treatment with PEI and conjugates. The level of hIL-12 expression was presented as the concentration of the protein (pg/mL) per seeded cells. In (c), each conjugate was compared to itself at the same C/P ratio in both cell lines. ^ap-value < 0.05, ^bp-value < 0.001 & ^cp-value < 0.0001, Conjugates were compared to unmodified PEI at the same C/P ratio (N = 3; error bars represent ± SD).

The level of hIL-12 production was also evaluated on 4T1 cells over-expressing LAT-1 transporter. The results revealed that the level of the protein expression elevated in all C/P ratios for all PEI conjugates. For instance, at the highest C/P ratio tested in this investigation (i.e. C/P = 8), PEI-LD 2% and 4% resulted in around 2.5 fold increase in the transfection efficiency. Meanwhile, the lowest degree of conjugation (i.e. PEI-LD 1%) resulted in a two folds enhancement in gene transfer efficiency. As shown in Fig. 6c, the level of hIL-12 production was illustrated for different conjugates on two cell lines at C/P ratios of 0.5–8. The results demonstrated that all conjugates have higher transfection efficiency on 4T1 cells compared with HepG2 cell lines at the same C/P ratios. For instance, PEI-LD 4% at all C/P ratios resulted in a 2.5 fold increase in the level of hIL-12 on 4T1 cell line compared with HepG2 cells. This transfection enhancement could be associated with the presence of LAT-1 transporters for cell entrance of PEI-LD conjugates. Since all polyplex formulations with unmodified PEI led to no increase in transfection efficiency on both cells, it could be concluded that the mechanism for cell entrance of unmodified PEI is similar in two cell lines. It has been reported that the main mechanism for unmodified PEI to enter the cells is adsorptive endocytosis resulting from the electrostatic interaction between the positively charged PEI complexes and the negatively-charged components on the cell membrane¹⁶. The conjugation of targeting moieties on the PEI polyplexes not only reduces the positive charge density leading to lower toxic effects, but also directs the polyplexes into the cells via specific mechanisms such as receptor/transporter mediated endocytosis. In order to exclude the probable effect of cell-induced toxicity by modified polymers on the efficiency of protein expression, the cell viability test was also conducted in a range of polymer concentrations. The results of cell viability tests (Fig. 5) revealed that the toxicity of modified polymers at the highest concentration (i.e.; 100 μg/mL) is about 20% lower. In other words, approximately 80% of cells are still alive after incubation by the modified polymers at the concentration of 100 μg/mL). The amount of modified polymer used to prepare C/P ratio = 4 is 80 μg/mL which showed no toxic effects on the cells. The elevated level of transfection efficiency on the cells lacking LAT-1 receptor is the result of lower zeta potential and lower chance of cell toxicity. Moreover, the enhanced gene transfer efficiency of PEI conjugates on the cells over-expressing LAT-1 transporters could be the result of transporter-mediated delivery.

In order to confirm the impact of LAT-1 transporters in the elevated gene transfer efficiency of PEI-LD conjugates, the transporters were competitively inhibited by the free LD at the concentration of 10^–3 mol/mL. The results of the competitive inhibition in 4T1 cell line indicated that the transfection efficiency of PEI-LD conjugates was not significantly different from that of unmodified PEI at the same C/P ratios (Fig. 7). The decreased level of hIL-12 by competitive inhibition of the LAT-1 transporter clearly demonstrated the role of the transporter in the elevated transfection efficiency of the PEI-LD conjugates.

Fig. 7.

Competitive inhibition of transfection efficiency of PEI and its conjugated derivatives complexed with pUMVC3-hIL12 in 4T1 cell lines at the concentration of 10^–3 mol/mL of free L-DOPA. ^ap-value < 0.05 and ^bp-value < 0.01, conjugated PEI in the absence of L-DOPA compared to the same derivative in the presence of L-DOPA (N = 3; error bars represent ± standard deviation).

Conclusion

In this study, L-DOPA-PEI derivatives were prepared and evaluated in terms of transfection efficiency and cytotoxicity. In conclusion, L-3, 4‐dihydroxyphenylalanine (L-DOPA)-conjugated HMW PEI could increase the delivery of hIL-12 plasmid into the cells over-expressing LAT-1 by up to 2.5 fold with reduced toxicity. Hence, decoration of PEI by targeting ligands could be considered as a promising approach to enhance gene delivery efficiency with low toxicity.

Author contributions

Conceptualization, F.A., A.D.; methodology, Z.T., M.K, B.K. and F.S.; software, Z.T., M.K., F.S. and A.D.; validation, A.D., F.A.; formal analysis, Z.T., M.K., F.A., A.D., B.K. and F.S.; investigation, Z.T., F.A. and A.D.; resources, B.K., F.A. and A.D. and.; data curation, Z.T., F.A. and A.D.; writing—original draft preparation, Z.T., F.A. and A.D.; writing—review and editing, Z.T., S.H.A, F.A. and A.D.; supervision, A.D.; project administration, F.A. and A.D.; funding acquisition, A.D. All authors have read and agreed to the published version of the manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author, Ali Dehshahri (A.D.), upon reasonable request.

Competing interests

The authors declare no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71798-1.