Interaction Kinetics of Peptide Lipids-Mediated Gene Delivery

doi:10.21203/rs.2.18224/v1

Download PDF

Research

Interaction Kinetics of Peptide Lipids-Mediated Gene Delivery

https://doi.org/10.21203/rs.2.18224/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 17 Oct, 2020

Read the published version in Journal of Nanobiotechnology →

You are reading this older preprint version

Read the latest preprint version →

Background: During the course of gene transfection, the interaction kinetics between liposomes and DNA is speculated to play very important role for blood stability, cellular uptake, DNA release and finally transfection efficiency.

Results: As cationic peptide liposomes exhibited great gene transfer activities both in vitro and in vivo, two peptide lipids, containing a tri-ornithine head (LOrn3) and a mono-ornithine head (LOrn1), were chosen to further clarify the process of liposome-mediated gene delivery in this study. The results show that the electrostatically-driven binding between DNA and liposomes reached nearly 100% at equilibrium, and high affinity of LOrn3 to DNA led to fast binding rate between them. The binding process between LOrn3 and DNA conformed to the kinetics equation: y = 1.663631 × exp(-0.003427x) + 6.278163. Compared to liposome LOrn1, the liposome LOrn3/DNA lipoplex exhibited a faster and more uniform uptake in Hela cells, as LOrn3 with a tri-ornithine peptide headgroup had a stronger interaction with the negatively charged cell membrane than LOrn1. The efficient endosomal escape of DNA from LOrn3 lipoplexes was facilitated by the acidity in late endosomes, resulting in broken carbamate bonds, as well as the “proton sponge effect” of the lipid.

Conclusions: The interaction kinetics is a key factor for DNA transfection efficiency. This work provided insights into peptide lipid-mediated DNA delivery that could guide the development of the next generation of delivery systems for gene therapeutics.

Nanoscience

Peptide lipids

Interaction kinetics

Uptake

Gene release

Gene delivery

Peptide-based cationic lipids are promising nonviral tools for nucleic acid delivery into cells, e.g., plasmids DNA for transfection and siRNA for gene silencing. They have shown to possess properties that are superior to those of other cationic lipids, such as good biodegradability, excellent biocompatibility, and the ability to target to cells [1–9]. The primary requirements for a successful peptide lipid vector are the ability to compact nucleic acid, to protect it against degradation, and to deliver it into the cell with efficiency and specificity. If the association of liposomes with nucleic acid is too weak, DNA or siRNA could not reach the target because that gene would be released into the blood in advance. But strong binding can lead to difficulties regarding the release of nucleic acid from endosomes into the nucleus [10]. Therefore, a perfect balance in the interaction between the liposomes and nucleic acid is required for the lipoplexes to behave as successful gene delivery vectors. In addition, liposomes/DNA lipoplexes must bind to the cell surface, enter cells by adsorption-mediated endocytosis, and accumulate in endosomes [11,12]. Finally, lipoplexes escape from the endosomes and release the nucleic acid for successful gene expression [13–15]. In the process of delivering nucleic acid by peptide liposome vectors, the interaction between them has a very important effect on cellular uptake, nucleic acid release, and DNA or siRNA escape from endosomes, which are necessary for efficient gene transfer. Without knowledge of the interaction of liposomes with gene and the limiting barriers involved, it will be difficult to take a rational approach to develop improved methods of gene transfer and to test specific hypotheses related to cellular and molecular mechanisms. However, systemic and thorough interaction research between peptide-based lipids and DNA has hardly been performed by far, and the effect of interaction on transfection efficiency hasn't been revealed.

See Fig. 1

To better understand the gene transport process and develop additional applications of peptide lipids, we here report on the use of 1,2-bis-myristyloxyamidopropyl tri-ornithine lipid (Orn3) for performing the research, and we used 1,2-bis-myristyloxyamidopropyl ornithine lipid (Orn1) as a control (their structures were shown in Fig. 1). Both were developed in our laboratory, and these lipids could efficiently transfer DNA and siRNA into tumor cells and tumors of mice, with little in vitro and in vivo toxicity [16].

We examined the kinetics of association and dissociation between lipids and DNA. The cellular uptake of the lipoplexes at the microscopic level was further analyzed. Furthermore, endosomal escape of DNA as mediated by lipoplexes was detected by live cell imaging of Hela cells to further clarify the mechanism of lipid-mediated delivery of DNA. As shown in Fig. 2, the peptide lipids self-assembled into liposomes, which could bind DNA by charge interactions (Fig. 2a). The lipid in liposome was composed of acid-responsive carbamate bond, lipoplexes could disassemble under disassembly pH (pH_dis) (Fig. 2b). After uptake by the cells, the lipoplexes could stimulate endosome escape due to proton buffering capacity. DNA was then released from lipoplexes under the endosomes environment (pH = 5.0~5.5), and efficient gene expression was obtained (Fig. 2c). In this process, we have found that the liposome LOrn3 with tri-peptide headgroup showed a significantly faster binding rate and higher affinity to DNA, and also showed higher efficiency for gene delivery. Hence, the revealed interaction kinetics of peptide lipids-mediated gene delivery in this study may serve guiding information in designing of gene carriers and studying mechanism of gene delivery.

See Fig. 2

Materials

The peptide lipids Orn1 and Orn3 were synthesized by our lab. The co-lipid 1,2-Dioleoyl-3-trimetry-lammonium-propane chloride salt (DOPE) was purchased from Sigma-Aldrich (USA). The plasmid pGFP-N1 was purchased from Promega Biotech Co. Ltd. (Beijing, China), and extracted in our lab. FAM-DNA was purchased from GenPharma company (China). The DNA intercalating dyes GelRed (10,000 × in water), heparin, the lipoosme intercalating dye N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-PE), hoechst and Cy5 Nucleic Acid Labeling Kit were purchased from Biyotime biotechnology company (China), respectively. Trypsin were purchased from Gibco company (USA). All other chemicals were purchased from Alading (Shanghai, China). All water used was purified using a Milli-Q Plus 185 water purification system (Millipore, USA), giving a resistivity greater than 18 MΩ.

Physicochemical properties of liposomes

The average particle sizes of liposomes were measured by dynamic light scattering (DLS, HORIBASZ-100, Japan). Twenty μL of lipoplexes were diluted to 1 mL with distilled water, all measurements were performed with freshly prepared liposomes at 25 °C and in triplicate.

Cell culture

The Hela cell line was purchased from Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China) and grown in Dulbecco's modified Eagle's medium (DMEM, Gibco, Inc., USA) media supplemented with 10% fetal bovine serum (Gibco, Inc.) and 1% antibiotic mixture. The cells were grown at 37℃ with 65% humidity.

DNA transfection

In vitro DNA transfection of liposomes/pGFP-N1 lipoplexes was measured against Hela cell. The cells were seeded at 5×10⁵ cells/well in 24-well plates. After 24 h, medium was replaced with serum-free medium, and lipoplexes in medium were added to each well and incubated for 4 h at 37 °C. The medium was then replaced with medium containing 10% FBS and 1% antibiotics, and the cultures were maintained at 37 °C under 5% CO₂. The expression of green fluorescent protein was measured by an inverted fluorescence microscope (Olympus IX71, Japan) and a flow cytometry (Becton-Dickinson, Heidelberg, Germany).

Agarose gel electrophoresis

The interaction of peptide liposomes with DNA was confirmed by gel retardation assay. The liposome was incubated with DNA for different time at room temperature with N/P ratios of 3:1 and 4:1 for Orn3 and Orn1. The final volume of micelle lipoplexes was adjusted to 20 µL, and then mixed with 6× loading buffer (2 μL). The mixture was loaded onto a 1.2 % agarose gel containing 3μL GelRed. Electrophoresis was carried out in 1× TAE running buffer at 90 V for 50 min, and DNA bands were visualised at UV light wavelength of 254 nm by a gel documentation unit (LongGene LG3000, China). The binding amounts of DNA by peptide liposomes (or DNA release rate) were calculated using software of gel documentation unit.

Steady-state fluorescence spectroscopic of DNA binded with liposomes

To make the GelRed labeled DNA, the DNA stock solutions and GelRed stock solutions were mixed at the DNA and GelRed weight ratio of 10:1 in the phosphate buffer and equilibrated for 15 min. To make the liposomes/DNA lipoplexes, the peptide liposomes Orn1 and DNA were mixed at 4:1 weight ratio, the ratio was 3:1 for Orn3 and DNA. To a GelRed-DNA mixture in a quartz cuvette were added the desired amounts of peptide liposomes stock solutions. The excitation wavelength of GelRed was 260 nm, the emission was 600 nm. The excitation slit and emission slit were fixed at 1 and 0.8 nm, respectively. The temperature was set at 25 °C. Recorded the fluorescence intensity of system for every 5–10 minutes.

Stopped-flow fluorescence spectroscopic of DNA binded with liposomes

The kinetic measurements were carried out using a stopped-flow fluorescence spectroscopic (SX-20MV, Applied Photophysics Ltd, UK). The concentration of both DNA and GelRed in the GelRed-DNA complex solution was, and 1mg/mL of the liposomes solution was used for stopped-flow kinetic study to obtain the charge ratio of 3:1 or 4:1. The excitation and emission monochromator were set to 480 and 600 nm, respectively. During the experiment, two separate syringes of the stopped-flow were filled up with DNA-GelRed complex and liposome solutions, and in each run, an equal volume of both solutions was injected at once into the sample chamber. The emission spectra were monitored continuously both before (t=0 s) and after the injection.

The release of DNA from lipoplexes with agarose gel electrophoresis

The lipoplexes were prepared with the method of DNA-binding assay above. The Orn1/DNA and Orn3/DNA lipoplexes were added to a given concentrations (0.05–2.0 µg/µL) of heparin dissolved in PBS and incubated for 30 min at room temperature [17]. The samples (20 µL) were electrophoresed on 1.2% agarose gel at different incubation time. Small amount of Orn1/DNA and LOrn3/DNA lipoplexes were incubated with pH 5.5 phosphate buffer for 1, 4, 12, 24, 36, 48, 60 and 72 h, respectively, and the resulting solution was assessed by agarose gel electrophoresis.

Cellular uptake using flow cytometry

Cellular uptake experiments were performed on Hela cell line. Orn1 and Orn3 Liposomes with the concentration range of 1.8 µg/mL were prepared (diluted by DMEM without FBS, pH 7.4), respectively. The liposome/FAM-DNA weight ratios were 4:1 and 3:1 for Orn1 and Orn3, respectively. Hela cells were seeded in 24-well plates at a density of 10⁵ cells/well and incubated in complete cell medium for 24 h. Then, the medium was replaced with 0.5 mL liposomes, and the plates were incubated at 37 °C for 4 h. Cells were washed with PBS, trypsinized, and resuspended in PBS at a concentration of 1×10⁶ cells/mL. Then that used Trypan Blue to quench FAM-DNA fluorescence outside of cells, a stock solution of 0.4% (w/w). Trypan Blue in water was prepared and added at 10% of the volume of the cell suspension immediately before measurement. Cells were detected and quantified by flow cytometry (Becton-Dickinson, Heidelberg, Germany). Data were processed using the Cflow Plus software (Becton-Dickinson) [18, 19].

Cellular uptake and DNA release using CLSM

Confocal laser scanning microscopy (CLSM) and TEM were used to investigate the intracellular transport of lipoplexes [20, 21]. Hoechst 33342, NBD-PE and Cy5-DNA were used here in order to detect the intracellular distribution of liposome/DNA lipoplexes. Orn1 (or Orn3), combined with DOPE (1:1 weight ratio), was mixed chloroform with NBD-PE, to achieve a final concentration of were prepared the day before the experiment, stored overnight at 4 °C, and sonicated immediately before use, liposome/NBD-PE volume ratio was 20:1 (concentration of liposome and NBD-PE both were 1mg/mL). Cy5-labeled DNA was prepared by use of the Label IT Cy5 Nucleic Acid Labeling Kit, according to the manufacturer's instructions. NBD-PE labeled liposome. Hela cells were seeded into 6-well plates, cells were transfected with liposome/DNA lipoplexes after 24 h. After 30 min, 2 h and 4 h of transfection, the treated cells were washed three times with 1 × PBS to remove residual free lipoplexes. Then 400 µL fresh DMEM containing Hoechst 33342 was added into each well to stain cells. Hoechst 33342 was used to stain cell nuclei. At last, the treated cells were imaged by CLSM (Olympus FV1000, Japan). For study of intracellular DNA release, LysoTracker Red was used to stain endosomes [22]. Cells were exposed to Orn3 liposome for 30 min, 2, and 4 h, cells were washed 3 times with PBS, fixed with 2.5% formaldehyde/2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at room temperature, and kept at 4 °C overnight. Then, the samples were washed several times in water, then dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in Spurr's low-viscosity medium. The samples were polymerized in a 60 °C oven for 2 days. Ultrathin sections were cut in a Leica Ultracut microtome, stained with lead citrate in a Leica EM stainer (Leica, EM FC7 UC7, USA), and examined in a transmission electron microscope (JEM-2100, Carl Zeiss, Japan).

Statistical analysis

All data were expressed as mean ± standard deviation (SD) for three independent tests. Differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by LSD's multiple comparison post hoc test. The results were considered statistically significant when p < 0.05 (^*, p < 0.05; ^**, p < 0.01;^*^**, p < 0.001).

Characterization of liposomes

Our previous study showed that peptide liposomes with tri-ornithine as the headgroup (LOrn3) could transfer DNA and DNA very efficiently into tumor cells, such as NCI-H460 and Hep-2 cells. Also, it was able to deliver combined DNAs against c-Myc and VEGF for silencing distinct oncogenic pathways in lung tumors of mice, with little in vitro and in vivo toxicity [16]. To develop further applications of peptide lipids for gene delivery, a comparison with the lipid LOrn1, with one ornithine as the headgroup, was used to obtain detailed information about the interaction between peptide lipids and genes and the intracellular transport process. The liposomes LOrn1 and LOrn3 were prepared by using the peptide lipids with DOPE as a co-lipid at a molar ratio of 1:1. Figure 2a shows that the sizes were around 80 nm for both liposomes LOrn1 and LOrn3 (Fig. 3a). And the Zeta potentials of LOrn1 and LOrn3 were 30 mV and 45 mV respectively (Fig. 3b), which ensured the stability of the liposomes. The morphological characteristics were visualized by transmission electron microscopy (TEM) (Fig. 3c). Images obtained from LOrn1 and LOrn3 liposomes revealed well-defined spheres structures, regularly shaped and uniformly distributed.

See Fig. 3

DNA transfection in vitro

To evaluate further transfection efficiency of the two liposomes, we used them to deliver the green fluorescent protein reporter gene (GFP) into Hela cells. The expression levels of GFP are shown in Figure 4a and 4b after the qualitative measurement of fluorescence and the quantitative determination of the fluorescent intensity. The N/P ratios had a great impact on DNA transfection, LOrn1 and LOrn3 possessed the highest transfection efficiency at N/P ratios of 4:1 and 3:1, respectively. And the expression of GFP of the LOrn3-treated cells was significantly stronger than that of the LOrn1-treated cells at the N/P ratios from 1:1 to 8:1 in Hela cells (Fig. 4a and b). Then, we investigated the ability of liposomes to compress DNA at N/P ratios from 0.5:1 to 8:1, findings in the gel retardation assay revealed that DNA binding amount increased with the N/P ratios increase. LOrn1 and LOrn3 completely bound DNA at N/P ratios more than 4:1 and 3:1, respectively (Fig. 4c and d). Meanwhile, the Zeta potentials of lipoplexes were detected, the results show that their Zeta potentials increased from negative to positive with the increase of N/P ratios (Fig. 4e). For LOrn1 and LOrn3, the values became positive just at N/P ratios of 4:1 and 3:1, respectively, corresponding with the N/P ratios of complete DNA compression, these results accorded well with the above gel retardation assay results. This study indicates that LOrn3 was more effective than LOrn1 in terms of the interaction with DNA.

See Fig. 4

Binding kinetics of liposome with DNA

To obtain detailed information about the interaction between liposomes and DNA, we utilized agarose gel electrophoresis and fluorescent resonance spectroscopy to examine the time-dependent interaction of cationic peptide liposomes with DNA. As shown in Fig. 5a and b, the binding amount of DNA significantly increased with time, and nearly 100% DNA binding was reached after 30 min and 24 min for LOrn1 or LOrn3 liposomes, respectively. The data also demonstrated that the binding speed between liposome LOrn3 and DNA was faster than that between LOrn1 and DNA. The interaction between liposomes and DNA labeled with a nucleic dye (GelRed) was also monitored by fluorescence spectroscopy [23, 24]. GelRed fluoresces strongly when bound to DNA, and the displacement of GelRed from DNA by liposomes results in a decrease in fluorescence intensity that correlates with the amount of liposome binding DNA. The results show that the displacement of GelRed from DNA by liposome LOrn3 was much faster than that by LOrn1 (Fig. 5c). Moreover, the fluorescence intensity of GelRed/DNA was lower for LOrn3 when the displacement reached the equilibrium. The results substantiate the fact that the DNA binding of liposomes is faster at the initial stage, and LOrn3 has a stronger DNA binding ability than LOrn1. To further investigate the binding process of the two liposomes and DNA, a stopped-flow fluorimeter (SX-20MV, UK) setup was employed to monitor the formation of liposome/DNA lipoplexes using the fluorescent probe GelRed. The data indicate that GelRed fluorescence decreased much more rapidly for LOrn3 than LOrn1 (Fig. 5d), and the K values of LOrn1 and LOrn3 were 0.102795 and 0.003472, respectively. By fitting the binding data, it was found that the binding process between liposomes and DNA conformed to the kinetics equations: y = 0.235141 × exp (-0.102795x) + 3.461248 and y = 1.663631 × exp (-0.003427x) + 6.278163 for LOrn1 and LOrn3, respectively. It means that the binding rate of LOrn3 and DNA was faster than that of LOrn1 and DNA. Then the binding affinity of liposomes to DNA was identified by using MicroScale Thermophoresis (MST) performed on the Monolith NT.115Pico system. In this method, the affinity of liposome to DNA is represented by K_d value, which is the liposome concentration required to bind 50% DNA [25, 26]. The dose response curves generated by titrating LOrn1 or LOrn3 liposomes to DNA solutions (Fig. 5e) show that the K_d values of LOrn1 or LOrn3 were 0.296 and 0.136 µM, respectively. Therefore, we speculate that high affinity of LOrn3 to DNA led to fast binding rate between them, and then resulting in more efficient transfection. Therefore, we further evaluated the effect of interaction kinetics between the liposomes and DNA on the transfection efficiency. The expression of GFP gradually increased with binding time, and then reached to the maximum values at about 24 min and 32 min for LOrn3 and LOrn1 liposomes, respectively (Fig. 5f and g). At this time the interaction between liposomes and DNA also reached equilibrium (Fig. 5b and c). We confirmed that the interaction kinetics is a key factor for DNA transfection efficiency.

See Fig. 5

Stability of lipoplexes in blood mimicked environment

Blood is rich in negatively charged macromolecules, such as serum protein and haparin, and these materials are known to dissociate DNA from lipoplexes by the interaction between liposomes and serum proteins [27–30]. Therefore, we first examined the effect of serum on the dissociation of lipoplexes, as shown in Fig. 6a and b, which depicts DNA release of lipoplexes in a medium with varying serum concentrations. A serum concentration of 10% had nearly no obvious effect on the stability of both lipoplexes, with a DNA release of about 2%. With increasing serum concentrations, the stability of both lipoplexes decreased, and the LOrn1/DNA lipoplex showed much more DNA dissociation than the LOrn3/DNA lipoplex. When the serum concentrations were greater than 25%, the release of DNA from the LOrn1/DNA lipoplexes increased to 20–35%; in contrast, the percentage of DNA released from the LOrn3/DNA lipoplexes under the same conditions was much lower. Our previous study had already revealed that lower concentrations of serum (5% or 10%) resulted in only a small reduction in transfection efficacy; with 20% serum, the transfection efficacy was slightly lowered [31]. Therefore, we hypothesize LOrn3 could well protect DNA in the lipoplexes from the interaction with serum when they are in the bloodstream.

See Fig. 6

Heparin sodium can competitively bind to liposomes from lipoplexes, which may lead to the dissociation of lipoplexes in blood. Therefore, the stability of lipoplexes was further examined by adding heparin sodium to simulate the blood environment. Figure 6c and d showed that the brightness and the area of the bands corresponding to free plasmid DNA increased with the increase in heparin concentration. Heparin sodium affected the stability of the LOrn1/DNA lipoplex to a much greater extent than the LOrn3/DNA lipoplex in the concentration range of 0.1–2.0 µg/µL. At a heparin concentration that is common in the blood (0.1 µg/µL), the release of DNA from the LOrn1/DNA lipoplex was 40%, and it was 20% for the LOrn3/DNA lipoplex. Therefore, the LOrn3/DNA lipoplex could show great advantages over LOrn1/DNA when used for in vivo transfection.

Cellular uptake of lipoplexes

Cellular uptake is required for successful transfection of genes. The cellular uptake of liposomes/FAM-DNA lipoplexes was first evaluated by FACS within 4 h. As displayed in Fig. 7a, the cellular uptake increased gradually with the transfection time; cellular uptake rates of LOrn1 and LOrn3 were about 73% and 94% after 4 h, respectively. LOrn3 showed a stronger interaction with the negatively charged cell membrane than LOrn1, so the LOrn3/DNA lipoplex could more easily cross the cell membrane, resulting in a higher cellular uptake rate compared with LOrn1/DNA. And we examined its morphological features at the ultra-structural level in Hela cells incubated with LOrn3/DNA lipoplexes for different periods of time (Fig. 7b). The majority of LOrn3/DNA lipoplexes were found within early endocytic structures (endosomes) and multi-vesicular bodies in the early phase of uptake (2 h). They were detected within lysosomes 4 h after exposure of cells to LOrn3/DNA lipoplexes.

To further explore the kinetics of LOrn3/DNA lipoplex uptake, cellular uptake of the liposome/DNA lipoplexes was also investigated by laser scanning confocal microscope (LSCM). As illustrated in the merged images (Fig. 7c), LOrn3/DNA lipoplexes displayed visible signals in the cytoplasm at 30 min post-transfection, but only a small amount of LOrn1/DNA lipoplexes were internalized into the cells. Though more LOrn1/DNA lipoplexes were localized in the cytoplasm at 2 and 4 h post-transfection, it was found that the fluorescent signals of LOrn3/DNA lipoplexes were distributed throughout the entire cytoplasm at 2 h post-transfection and mainly gathered around the nuclei at 4 h post-transfection. Compared to liposome LOrn1, liposome LOrn3/DNA lipoplexes exhibited a faster and more uniform uptake in Hela cells. These results also indicate that DNA delivered with LOrn3 liposomes accumulated in a compartment having biochemical and morphological characteristics of both early and late endosomes and that matured slowly. And combined with the interaction data of liposome and DNA (Fig. 5), it's found that the more the liposome-DNA interaction was, the higher the cellular uptake of liposome/DNA lipoplex was.

See Fig. 7

Dissociation kinetics of liposome and DNA

After the assembly with DNA, the stimuli-responsive features and DNA dissociation behavior of the lipoplexes were tested in vitro in a simulated intracellular microenvironment (pH 5–5.5) using a gel retardation assay [32, 33]. As can be seen in Figure 8a and b, the DNA dissociated from lipoplexes was time-dependent at pH 5.5, and the DNA dissociation rate of the LOrn3/DNA lipoplex was faster than that of the LOrn1/DNA lipoplex. Seventy percent of DNA could dissociate from LOrn3/DNA lipoplexes and only 51% from LOrn1/DNA lipoplexes at 72 h. In contrast, little DNA dissociated from the two lipoplexes at the similar time point (8% at 72 h) at pH 7.0. The pH-induced structural destruction of lipoplexes contributed to the DNA release by exposing carbamate bonds in the lipids to the reductive environment for cleavage (Additional file 1: Figure S1).

See Fig. 8

A major intracellular barrier for DNA delivery is endosomal entrapment followed by trafficking and lysosomal degradation or exocytosis [34, 35]. The escape of DNA from endosomes into the cytosol is thought to be a rate-limiting step for many delivery approaches [36]. The lipoplexes are designed to facilitate late endosomal escape of DNA by synergistic effects of the proton sponge effect [37, 38] and the degradation of the peptide lipid LOrn1 or LOrn3 by carbamate bond breaks in endosomes. To assess intracellular DNA release, confocal microscopy was used to investigate the distribution of FAM-labeled DNA (green) in lipoplexes and LysoTracker Red labeled endosomes against Hela cells. As shown in Figure 8c, we observed that DNA was gradually released from the both of lipoplexes over time. For LOrn3/DNA lipoplexes, most FAM signals of the LOrn3 lipoplexes were merged in endosomes at 12 h, as shown by the yellow color. Much DNA could be released from endosomes at 24 h after the transfection, and at 48 h, strong green fluorescence in the images for LOrn3/DNA lipoplexes showed an obvious release of DNA from endosomes (indicated with white arrows).

The co-localization ratio (%) is a commonly used index to measure DNA release [39, 40], a low co-localization ratio means quick DNA release from the endosomes and a better distribution of the DNA in cytoplasm. The results show that the co-localization ratio gradually decreased with time; they were 49% and 58% at 48 h for LOrn3 and LOrn1, respectively (Fig. 8d). According to the results of the lipoplex dissociation study in a simulated tumor microenvironment (Fig. 8b), the response to the acidic environment was probably caused by the break of carbamate bonds in the lipids. In addtition, DNA release of LOrn3/DNA lipoplex was more than that of LOrn1/DNA lipoplexe at 48 h, this was consistent with the result in Fig. 8b. Although LOrn1 and LOrn3 have a similar chemical constitution, LOrn3, containing more amino groups, promoted not only cellular internalization [41] but also displayed a higher proton buffering capacity. Therefore, LOrn3 lipoplexes showed an enhanced capability in endosomal escape and DNA release compared to LOrn1 lipoplexes. That also explains the reason why LOrn3 had more efficient transfection than LOrn1. Accordingly, FRET was utilized to further confirm the intracellular disassembly process of DNA from the LOrn3/DNA lipoplexes in Hela cells, by using Rhodamine-DNA and NBD-PE-LOrn3 liposome to constitute a Rhodamine/NBD FRET system (acceptor/donor) [42]. After the Hela cells were transfected with lipoplexes for 4 h, they were incubated for 2, 4, 6, 8, 12, 24, 36, and 48 h. Figure 8e shows the fluorescence emission intensity of Rhodamine-labeled DNA decreased with time. The data indicate that less resonance energy was transferred from donor to acceptor due to lipoplex dissociation in Hela cells, and much of the DNA was released from lipoplexes at 48 h after transfection.

Together, these results strongly demonstrated that both of the lipoplexes could exist in a comparatively stable structure under normal physiological conditions (pH 7.0), while they rapidly release a significant amount of DNA due to the destabilization resulting from the acidic microenvironment in endosomes or lysosomes of tumor cells (pH 5.5) [43, 44].

Our study provides insights into the interaction kinetics, cellular uptake, and dissociation of peptide liposome-mediated gene delivery. It highlights similarities and differences between liposomes with either tri-ornithine or mono-ornithine headgroups. We demonstrate that the peptide lipid with three ornithine headgroup (LOrn3) had a strong binding ability to DNA. Moreover, LOrn3/DNA lipoplexes exhibited excellent intracellular stability, effective cellular uptake, lysosomal evasion, and intracellular gene release. Most importantly, we have illustrated the relationship between interaction kinetics and transfection efficiency. In summary, the study proposes a theoretical framework for strategies to improve gene delivery systems.

Authors´ contributions

YNZ and SBZ conceived and designed the study, discussed the results and drafted the manuscript. YYD and YNC performed the cell experiments and analyzed the data. YX contributed to the liposomes preparation for size and zeta measurements. STG and HYC contributed to a part of data analysis work. DFZ contributed to the editing of the manuscript.

Acknowledgements

The authors thank for the financial support of National Science Foundation of China (NSFC. 21606041, 21776044, 21908019). Liaoning Provincial Natural Science Foundation ().

Availability of data and materials

All data generated or analyzed during this research are included in this article.

Consent for publication

Not applicable within this study.

Ethics approval and consent to participate

Not applicable within this study.

Competing interests

The authors declare that they have no competing interests.

Author details

¹ Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Sciences, Dalian Minzu University, Dalian 116600, China. ² Key Laboratory of Functional Polymer Materials of Ministry of Education, State Key Laboratory of Medicinal Chemical Biology and Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin, 300071, China

Wan Y, Dai W, Nevagi RJ, Toth I, Moyle PM. Multifunctional peptide-lipid nanocomplexes for efficient targeted delivery of DNA and siRNA into breast cancer cells. Acta Biomaterialia 2017; 59: 257-
Suga T, Fuchigami Y, Hagimori M, Kawakami S. Ligand peptide-grafted PEGylated liposomes using HER2 targeted peptide-lipid derivatives for targeted delivery in breast cancer cells: the effect of serine-glycine repeated peptides as a spacer. J. Pharmaceut. 2017; 521: 361-364.
Niu J, Chu Y, Huang YF, Chong YS, Jiang ZH, Mao ZW, Peng LH, Gao JQ. Transdermal gene delivery by functional peptide-conjugated cationic gold nanoparticle reverses the progression and metastasis of cutaneous melanoma. ACS Appl. Mater. Interfaces 2017; 9: 9388-
Jayant RD, Sosa D, Kaushik A, Atluri V, Vashist A, Tomitaka A, Nair M. Current status of non-viral gene therapy for CNS disorders. Expert Opin. Drug Del. 2016; 10: 1433-1445.
Ashley CE, Carnes EC, Epler KE, Padilla DP, Phillips GK, Castillo RE, Wilkinson DC, Wilkinson BS, Burgard CA, Kalinich RM, Townson JL, Chackerian B, Willman CL, Peabody DS, Wharton W, Brinker CJ. Delivery of small interfering RNA by peptide-targeted mesoporous silica nanoparticle-supported lipid bilayer ACS Nano 2012; 3: 2174-2188.
Tagalakis AD, Lee DHD, Bienemann AS, Zhou HY, Munye MM, Saraiva L, McCarthy D, Du ZX, Vink CA, Maeshima R, White EA, Gustafsson K, Hart SL. Multifunctional, self-assembling anionic peptide-lipid nanocomplexes for targeted siRNA delivery. Biomaterials 2014; 35: 8406-8415.
Nie TQ, He ZY, Zhou Y, Zhu JC, Chen KT, Liu LX, Leong KW, Mao HQ, Chen YM. Surface coating approach to overcome mucosal entrapment of DNA nanoparticles for oral gene delivery of glucagon-like peptide 1. ACS Appl. Mater. Interfaces 2019; 11: 29593-29603.
Writer M, Kyrtatos PG, Bienemann AS, Pugh JA, Lowe AS, Villegas-Llerena C, Kenny GD, White EA, Gill SS, McLeod CW, Lythgoe MF, Hart SL. Lipid peptide nanocomplexes for gene delivery and magnetic resonance imaging in the brain. Control. Release 2012; 2: 340-348.
Ganguly P, Breen A, Pillai SC. Toxicity of nanomaterials: exposure, pathways, assessment and recent advances. ACS Biomater. Sci. Eng. 2018; 4: 2237-2275.
Gilleron J, Querbes W, Zeigerer A, Borodovsky A, Marsico G, Schubert U, Manygoats K, Seifert S, Andree C, Stöter M, Epstein-Barash H, Zhang L, Koteliansky V, Fitzgerald K, Fava E, Bickle M, Kalaidzidis Y, Akinc A, Maier M, Zerial M. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Biotechnol. 2013; 7: 638-650.
Gao P, Pan W, Li N, Tang B. Boosting cancer therapy with organelle-targeted nanomaterials. ACS Appl. Mater. Interfaces 2019; 11: 26529-26558.
Hanzlíková M, Ruponen M, Galli E, Raasmaja A, Aseyev V, Tenhu H, Urtti A, Yliperttula M. Mechanisms of polyethylenimine-mediated DNA delivery: free carrier Helps to overcome the barrier of cell-surface glycosaminoglycans. Gene Med. 2011; 13: 402-409.
Mihaila R, Ruhela D, Keough E, Cherkaev E, Chang S, Galinski B, Bartz R, Brown D, Howell B, Cunningham JJ. Mathematical Modeling: A Tool for Optimization of Lipid Nanoparticle-Mediated Delivery of siRNA. Ther-Nucl. Acids 2017; 16: 246-255.
Rehman Z, Hoekstra D, Zuhorn IS. On the mechanism of polyplex-and lipoplex-mediated delivery of nucleic acids: real-time visualization of transient membrane destabilization without endosomal lysis. ACS Nano 2013; 7: 3767-3777.
Nguyen J, Szoka FC. Nucleic acid delivery: the missing pieces of the puzzle? Acc Chem. Res. 2012; 45: 1153-1162.
Zhao YN, Zhang SB, Zhang Y, Cui SH, Chen HY, Zhi DF, Zhen YH, Zhang SF, Huang L. Tri-peptide cationic lipids for gene delivery. Mater. Chem. B 2015; 3: 119-126.
Takahashi T, Harada A, Emi N, Kono K. Preparation of efficient gene carriers using a polyamidoamine dendron-bearing lipid: improvement of serum resistance. Bioconjugate Chem. 2005; 16: 1160-1165.
Zhao YN, Liu A, Du YY, Cao YN, Zhang EX, Zhou Q, Hai H, Zhen YH, Zhang SB. Effects of sucrose ester structures on liposome mediated gene delivery. Acta Biomaterialia 2018; 72: 278-286.
Miteva M, Kirkbride KC, Kilchrist KV, Werfel TA, Li H, Nelson CE, Gupta MK, Giorgio TD, Duvall CL. Tuning PEGylation of mixed micelles to overcome intracellular and systemic siRNA delivery barriers. Biomaterials 2015; 38: 97-107.
Park K, Yang JA, Lee MY, Lee H, Hahn SK. Reducible hyaluronic acid-siRNA conjugate for target specific gene silencing. Bioconjugate Chem. 2013; 24: 1201-1209.
Zhang J, Shrivastava S, Cleveland RO, Rabbitts TH. Lipid-mRNA nanoparticle designed to enhance intracellular delivery mediated by shock waves. ACS Appl. Mater. Interfaces 2019; 11: 10481-
Gujrati M, Shin MTA, Jin E, Sun Y, Lu ZR. Multifunctional cationic lipid-based nanoparticles facilitate endosomal escape and reduction-triggered cytosolic siRNA release. Pharm. 2014; 11: 2734-2744.
Madeira C, Loura LMS, Aires-Barros MR, Fedorov A, Prieto M. Characterization of DNA/Lipid complexes by fluorescence resonance energy transfer. J. 2003; 85: 3106-3119.
Grigsby CL, Ho YP, Leong KW. Understanding nonviral nucleic acid delivery with quantum dot-FRET nanosensors. Nanomedicine 2012; 4: 565-
Schuette CG, Hatsuzawa K, Margittai M, Stein A, Riedel D, Küster P, King M, Seidel C, Jahn R. Determinants of liposome fusion mediated by synaptic SNARE proteins. Proc. Natl. Acad. Sci. USA 2014: 2858-
Marsden HR, Korobko A, Zheng TT, Voskuhl J, Kros A. Controlled liposome fusion mediated by SNARE protein mimics. Biomater. Sci. 2013; 1: 1046-
Moret I, Peris JE, Guillem VM, Benet M, Revert F, Dasi F, Crespo A, Aliño SF. Stability of PEI-DNA and DOTAP-DNA complexes: effect of alkaline pH, heparin and serum. Control. Release 2001; 76: 169-181.
Kennedy MT, Pozharski EV, Rakhmanova VA, MacDonald RC. Factors governing the assembly of cationic phospholipid-DNA complexes. J. 2000; 78: 1620-1633.
Jones CH, Chen C, Ravikrishnan A, Rane S, Pfeifer BA. Overcoming nonviral gene delivery barriers: perspective and futur. Pharm. 2013; 11: 4082-4098.
Li S, Tseng WC, Stolz DB, Wu SP, Watkins SC, Huang L. Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection. Gene Ther. 1999; 6: 585-
Zhao YN, Zhu J, Zhou HJ, Guo X, Tian T, Cui SH, Zhen YH, Zhang SB, Xu YH. Sucrose ester based cationic liposomes as effective non-viral gene vectors for gene delivery. Colloids and Surfaces B: Biointerfaces 2016; 145: 454-461.
Zhu J, Qiao MX, Wang Q, Ye YQ, Ba S, Ma JJ, Hu HY, Zhao XL, Chen DW. Dual-responsive polyplexes with enhanced disassembly and endosomal escape for efficient delivery of siRNA. Biomaterials 2018; 162: 47-59.
Hong BJ, Chipre AJ, Nguyen ST. Acid-degradable polymer-caged lipoplex (PCL) platform for siRNA delivery: facile cellular triggered release of siRNA. Am. Chem. Soc. 2013; 135: 17655-17658.
Yang X, Fan B, Gao W, Li LP, Li TT, Sun JH, Peng XY, Li XY, Wang ZJ, Wang BQ, Zhang RP, Xie J. Enhanced endosomal escape by photothermal activation for improved small interfering RNA delivery and antitumor effect. J. Nanomedicine 2018; 13: 4333-4344.
Zhou ZW, Zhang QY, Zhang MH, Li HP, Chen G, Qian CG, Oupicky D, Sun MJ. ATP-activated decrosslinking and charge-reversal vectors for siRNA delivery and cancer therapy. Theranostics 2018; 17: 4604-4619.
Dominska M, Dykxhoorn DM. Breaking down the barriers: siRNA delivery and endosome escape. Cell Sci. 2010; 123: 1183-1189.
Ullah I, Chung K, Beloor J, Kim J, Cho M, Kim N. Trileucine residues in a ligand-CPP-based siRNA delivery platform improve endosomal escape of siRNA. Drug Target. 2017; 4: 320-329.
Forrest ML, Pack DW. On the kinetics of polyplex endocytic trafficking: implications for gene delivery vector design. Ther. 2002; 1: 57-66.
Han SC, Cheng Q, Wu YD, Zhou JH, Long XW, Wei T, Huang YY, Zheng SQ, Zhang JH, Deng LD, Wang XX, Liang XJ, Cao HQ, Liang ZC, Dong AJ. Effects of hydrphobic core components in amphiphilic PDMAEMA nanoparticles on siRNA delivery. Biomaterials 2015; 48: 45-55.
Du LL, Wang CR, Meng LW, Cheng Q, Zhou JH, Wang XX, Zhao DY, Zhang JH, Deng LD, Liang ZC, Dong AJ, Cao HQ. The study of relationships between pKa value and siRNA delivery efficiency based on tri-block copolymers. Biomaterials 2018; 176: 84-93.
Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, Suqiura Y. Arginine-rich peptides: an abundant source of membrane-permbeable peptides having potential as carriers for intracellular protein delivery. Biol. Chem. 2001; 8: 5836-5840.
Grigsby CL, Ho YP, Leong KW. Understanding nonviral nucleic acid delivery with quantum dot-FRET nanosensors. Nanomedicine 2012; 7: 565-577.
Kongkatigumjorn N, Smith SA, Chen M, Fang K, Yang SL, Gillies ER, Johnston APR, Such GK. Controlling endosomal escape using pH-responsive nanoparticles with tunable disassembly. ACS Appl. Nano Mater. 2018; 7: 3164-3173.
Wang C, Zhao T, Li Y, Huang G, White MA, Gao J. Investigation of endosome and lysosome biology by ultra pH-sensitive nanoprobes. Drug Deliver. Rev. 2017; 113: 87-96.

Download PDF

Journal Publication

published 17 Oct, 2020

Read the published version in Journal of Nanobiotechnology →

Editorial decision: Major revision
11 Feb, 2020
Review #2 received at journal
25 Jan, 2020
Reviewer #2 agreed at journal
13 Jan, 2020
Reviewer #3 agreed at journal
13 Jan, 2020
Review #1 received at journal
13 Jan, 2020
Reviewer #1 agreed at journal
10 Jan, 2020
Reviewers invited by journal
16 Dec, 2019
Editor invited by journal
02 Dec, 2019
Editor assigned by journal
02 Dec, 2019
First submitted to journal
01 Dec, 2019
Submission checks completed at journal
01 Dec, 2019

You are reading this older preprint version

Read the latest preprint version →

Interaction Kinetics of Peptide Lipids-Mediated Gene Delivery

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Results and discussion

Conclusions

Declarations

References

Status:

Journal Publication

Version 1