Preparation of cationic carbon dots and related gene delivery methods and systems

Abstract

The present invention relates to methods for preparing carbon-based nanoparticles, also known as carbon quantum dots or carbon dots (CDs), in particular cationic carbon dots (CCDs), by incubating (1) CDs obtained from the hydrothermal treatment of an amine-free aqueous solution of citric acid with (2) polyethylenimine (PEI) (3) using stirring. The invention further relates to the use of said CCDs in the generation of gene delivery and gene transfection systems for the production of retroviral vectors.

Description

[0001] DESCRIPTION

[0002] Preparation of cationic carbon dots v methods v associated gene delivery systems

[0003] FIELD OF INVENTION

[0004] The invention relates to gene delivery methods and systems and their uses, including the synthesis of carbon-based nanoparticles, also known as carbon quantum dots or carbon dots, particularly cationic carbon dots, for such gene delivery methods or systems.

[0005] BACKGROUND OF THE INVENTION

[0006] Cell transfection efficiency is a limiting factor for many biotechnological assays that require high gene transfer rates. Gene delivery strategies are generally classified according to their need to integrate into the cell genome for expression, which depends on the nature of the exogenous DNA construct (integrative or non-integrative) and the nature of the vector that allows entry into the target cell (viral or non-viral). These characteristics determine the efficiency, reproducibility, and safety (with respect to potential cell damage) of the method. Due to their exceptional infectiousness, virus-based vectors typically display excellent gene transfection capabilities, although they can induce cytopathic effects or random mutagenesis when integrative constructs are used, so their use tends to be avoided in biomedical applications.

[0007] For the introduction of nonviral vectors, a delivery method is required that allows the DNA vector to enter the cell for expression. Despite their advantages in terms of biological safety for cells, nonviral vectors have not been widely used due to their relatively low efficiency in gene delivery. There are different methodologies that can be classified into physical methods and chemical / carrier-based methods.

[0008] Physical methods include electroporation, particle bombardment, sonoporation, photoporation, and magnetofection, which use electrical pulses, force, sound, light, and magnetic fields to increase the permeability of cell membranes and allow genetic material to enter cells. However, these physical methods typically cause a high rate of cell death during the process and greatly affect their phenotype, which could affect downstream processes and increase the risk of artifactual results. However, there have been significant advances with specific strategies such as photoporation or electroporation, among others.

[0009] The alternative is to use synthetic or natural biocompatible materials as carriers to deliver genetic material that can enter cells by endocytosis. The most widely used nonviral vectors based on chemical carriers are liposomes and polymers. Nonviral liposome-based vectors use liposomes to facilitate gene delivery through the formation of lipoplexes. Lipoplexes form spontaneously when negatively charged DNA comes into contact with positively charged liposomes, and much progress has been made to improve transfection efficiency and overcome the toxicity of cationic lipids; even commercial formulations based on Lipofectamine reagents are commonly used in cell biology with varying degrees of efficacy and toxicity. Nonviral polymer-based vectors use polymers to interact with DNA and form polyplexes.Polyethyleneimine (PEI) is the most frequently used cationic polymer as a transfection reagent. It is positively charged and can form complexes with negatively charged nucleic acids, such as DNA or RNA. These complexes can efficiently deliver nucleic acids into cells by interacting with the cell membrane and facilitating their internalization. Various forms of PEI, considering their molecular weight and structure, have been used for gene delivery. The most common is the 25 kDa PEI, with either a branched or linear structure. The linear 25 kDa form is the favorite among most cell biologists, suggesting a better balance between DNA transfection efficiency and acceptable cellular toxicity.

[0010] Carbon dots (CDs), also known as carbon quantum dots, are nanometer-scale carbon-based materials with unique optical and chemical properties. They are primarily generated from carbonaceous sources and have been widely investigated for diverse applications, such as gene transfection, drug delivery, and cellular imaging. CDs can be functionalized to enhance their interactions with cells and exhibit strong biocompatibility. It has been reported that when CDs have positive surface charges from amino groups incorporated during their synthesis, they can bind to negatively charged DNA by electrostatic interactions to form CD-DNA complexes that can then penetrate cell membranes through mechanisms such as phagocytosis, endocytosis, and micropinocytosis.However, gene delivery efficiency has not been extensively analyzed in these reports, and two main strategies have been used to improve the binding efficiency, obtaining more positively charged CDs. Either (1) by using positively charged molecules as precursors, such as polyethyleneimine (PEI), polyethylene diamine (PAMAM), chitosan, polylysine-L (PLL), and pentaethylenehexamine (PEHA) for CD synthesis [1], or (2) through a two-step method, where first CDs containing -COOH and hydroxyl (-OH) groups on their surface are generated by different synthetic approaches, such as hydrothermal, pyrolytic, or microwave-assisted methods, resulting in a high density of functional groups, and then high-density -NH2 compounds are grafted onto the surface of CDs via amide reaction or electrostatic attraction, generating positively charged cationic CDs (CCDs) [2],.

[0011] The present invention relates to the generation of an efficient gene delivery method based on linear PEI-coated CCDs through electrostatic interactions. This method not only significantly improves cell transfection compared to commercial PEI polyplexes, but also enables simple and rapid preparation of the CCDs.

[0012] Although electrostatic interaction is a relatively weak force, the assembly of cationic carbon dots (CCDs) by this approach has the advantage of being simpler than the other methods described. However, its use has been limited to a few studies using PEI as a coating agent, not related to gene delivery, but rather to the detection of traces of chromium (Cr) in environmental water samples [3] or to the efficient imaging of specific cancer cells [4].

[0013] As mentioned, a combination of carbon dots and PEI has been employed for cell transfection in some studies, reporting some efficiency in gene delivery (reviewed in [5]), with very few studies showing significant improvements compared to commercial lipoplexes or polyplexes [6]. Reported strategies for CCD generation have focused on a one-step method using branched PEI together with a carbon precursor molecule for CCD synthesis, and only one study has been reported where a PEI variant (alkylated PEI, 2 kDa) was grafted after carbon dot synthesis via an amide reaction [7].

[0014] The present invention relates to the generation of an efficient gene delivery method based on linear PEI-coated CCDs via electrostatic interactions. This method not only significantly improves cell transfection compared to commercial PEI polyplexes, but also allows for simple and rapid preparation of the CCDs, enabling their use in a wide range of biological applications without relying on specialized chemical syntheses. Furthermore, it allows combination with different PEI ratios, enabling optimization for specific plasmids / genes, since the optimal DNA / PEI interaction depends on the nitrogen / phosphate (N / P) ratio, which varies depending on the plasmid / DNA length and the specific cell type.

[0015] SUMMARY OF THE INVENTION

[0016] Gene delivery is a complex process that faces several challenges when attempting to efficiently and safely incorporate genetic material into target cells. Some of the key challenges include not only efficient cellular uptake and endosomal egress to ensure the genetic material can exert its effect, but also minimizing the toxicity of the delivery system, which is crucial for safe gene delivery. Of utmost importance, if gene delivery systems are intended for biomedical or clinical applications, they must be scalable, easy, and affordable to manufacture to meet demand. Presented here is an efficient gene delivery method using a combination of PEI-coated carbon dots via electrostatic bonding, allowing for the easy generation of cationic carbon dots.This is a biofunctional approach to generating optimal cationic carbon dots (CCDs) that can be scaled to meet specific transfection demands. CCDs improve cell viability and increase transfection efficiency fourfold compared to standard PEI polyplexes.

[0017] A first aspect of the present invention relates to a method for preparing cationic carbon dots (CCDs) comprising incubating the CDs with PEI under stirring. In a preferred embodiment of the method for preparing cationic carbon dots (CCDs), the CDs used for incubation with PEI have a spherical morphology, with an average size of less than 5 nm in diameter and a hydrodynamic diameter (HDD) of less than 10 nm.

[0018] In a preferred embodiment of the method for the preparation of cationic carbon dots (CCDs), the CDs used for incubation with PEI are those obtained from the hydrothermal treatment of an aqueous solution of citric acid.

[0019] In a preferred embodiment of the method for the preparation of cationic carbon dots (CCDs), 25 kDa linear PEI is used for incubation with the CDs.

[0020] In a preferred embodiment of the method for the preparation of cationic carbon dots (CCDs), the incubation with shaking is carried out for 3 hours at room temperature (25 °C).

[0021] In another preferred embodiment of the method for preparing cationic carbon dots (CCDs), the CDs for incubation with PEI are added as an aqueous solution with a CD concentration ranging from 0.5 g / L to 0.025 g / L. In a more preferred embodiment, the CDs for incubation with PEI are added as an aqueous solution with a CD concentration ranging from 0.1 g / L to 0.025 g / L. In an even more preferred embodiment, the CDs for incubation with PEI are added as an aqueous solution with a CD concentration of 0.05 g / L.

[0022] In another preferred embodiment, the PEI for incubation with the CDs is added obtaining a mass ratio CD (pg) : PEI (pg) ranging from 10:3 to 200:3. In a more preferred embodiment, the PEI for incubation with the CDs is added obtaining a mass ratio CD (pg) : PEI (pg) ranging from 10:3 to 40:3. In an even more preferred embodiment, the PEI for incubation with the CDs is added obtaining a mass ratio CD (pg) : PEI (pg) of 20:3.

[0023] A second aspect of the present invention relates to the CCDs resulting from a preparation method according to the first aspect of the present invention.

[0024] In a preferred embodiment, the CCDs resulting from a preparation method according to the first aspect of the present invention have a spherical morphology, with an average size greater than 50 nm in diameter and an average hydrodynamic diameter of approximately 100 nm, 30 minutes after preparation of the CCDs.

[0025] A third aspect of the present invention relates to a gene delivery system comprising DNA combined with CCDs resulting from a preparation method according to the first aspect of the present invention.

[0026] In a preferred embodiment of a gene delivery system comprising DNA combined with CCDs resulting from a preparation method according to the first aspect of the present invention, the DNA to be combined with the CCDs is added obtaining a mass ratio CD (pg) : PEI (pg) : DNA (pg) ranging from 20:3:0.2 to 20:3:5. In a more preferred embodiment, the DNA to be combined with the CCDs is added obtaining a mass ratio CD (pg) : PEI (pg) : DNA (pg) ranging from 20:3:0.5 to 20:3:5. In an even more preferred embodiment, the DNA to be combined with the CCDs is added obtaining a mass ratio CD (pg) : PEI (pg) : DNA (pg) ranging from 20:3:1 to 20:3:5. In an even more preferred embodiment, the DNA to be combined with the CCDs is added obtaining a mass ratio CD (pg): PEI (pg): DNA (pg) of 20:3:1.

[0027] A fourth aspect of the present invention relates to a gene delivery method comprising incubating the cells to be genetically modified with a gene delivery system according to the third aspect of the present invention.

[0028] The generated CCDs enabled a challenging transfection protocol to produce retroviral vectors by co-transfecting three different plasmids into packaging cells, showing not only high efficiency, but also functionality in gene delivery, proven by the ability to produce infective retroviral particles.

[0029] A fifth aspect of the present invention relates to a gene transfection system for producing retroviral vectors comprising a gene delivery system according to the fourth aspect of the present invention, wherein the DNA includes a plasmid encoding the transgene in combination with a packaging plasmid and an envelope plasmid. In a preferred embodiment of a gene transfection system for producing retroviral vectors, twice the amount of plasmid encoding the transgene is combined with the packaging and envelope plasmids, resulting in a molecule:molecule ratio of 2:1:1.

[0030] In a preferred embodiment of a gene transfection system for producing retroviral vectors, the bicistronic plasmid pMX-GFP-Sox15 is used as the plasmid encoding the transgene, the plasmid pCMV-GAG-POL is used as the plasmid encoding the packaging proteins, and the plasmid pCMV-VSV is used as the plasmid encoding the envelope proteins.

[0031] A sixth aspect of the present invention relates to a gene transfection method comprising a gene transfection system according to the fifth aspect of the present invention.

[0032] In a preferred embodiment of a gene transfection method for producing retroviral vectors, HEK293T cells are (1) cultured in high glucose DMEM (with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin); (2) seeded at 70% cell confluence; and (3) transfected with a gene transfection system according to the sixth aspect of the present invention; 24 hours after transfection, the supernatant is collected and filtered.

[0033] In a preferred embodiment of a gene transfection method for producing retroviral vectors, primary somatic stromal cell lines MenSC (also called MnSC) are (1) cultured in DMEM-F12 containing 10% FBS, IxNEAA, 1xL-Glutamine, penicillin and streptomycin; (2) seeded at 70% cell confluence; and (3) transfected with a gene transfection system according to the sixth aspect of the present invention; 24 hours after transfection, the supernatant is collected and filtered.

[0034] BRIEF DESCRIPTION OF THE FIGURES

[0035] Figure 1. Schematic representation of a pMXs-based vector, a bicistronic retroviral vector that allows the expression of human SOX15 and GFP. (Source: Figure 2. (A) HR-TEM images of CDs generated from the citric acid reaction at 200 °C, shown at two magnifications (scale bar 5 nm). (B) Frequency histogram of DLS measurements of the mean hydrodynamic diameter (nm) found in CD suspensions. UV / Vis (C) and fluorescence (D) spectra of the CDs generated from citric acid.

[0036] Figure 3. Relative cell viability of HEK293T cells after 24 h of incubation with different concentrations of CDs (n=5, mean ± SD). Statistical significance was determined using Student's t test: *P<0.05, ***P<0.001.

[0037] Figure 4. (A) Schematic of CCD preparation and cell transfection. (B) Transfection efficiency, measured as % of GFP-positive cells by flow cytometry, of HEK293T cells seeded in p6-well plates and transfected with CCDs generated from incubating CDs with PEI at different mass ratios and combined with a fixed amount of DNA. PEI at a 3:1 mass ratio with DNA was used as a control (orange) (n=7, mean±SD. Statistical significance was determined by Student’s t-test. ***P<0.001). (C) Representative fluorescence microscopy images of HEK293T cells transfected with CCDs (20:3 mass ratio) or PEI (scale bar 100 µm). (D) Transfection efficiency, measured as the % of GFP-positive cells measured by flow cytometry, of MenSC cells seeded in p6-well plates and transfected with CCDs (mass ratio 20:3).(E) Representative fluorescence microscopy images of MenSC cells transfected with CCDs or PEI (scale bar 100 pm).

[0038] Figure 5. (A) TEM images of a 50 g / L CCD suspension prepared as described by 3 h of mixing CDs with PEI at an optimized mass ratio of 20:3 (scale bar 50 nm), 30 min after preparation. (B) Frequency histogram of DLS measurements of the mean hydrodynamic diameter (nm) found in CCD suspensions 30 min after preparation.

[0039] Figure 6. (A, C, E) TEM images of a 50 g / L CCD suspension prepared as described by 3 hours of mixing CDs with PEI at an optimized mass ratio of 20:3 (scale bar 50 nm), 2 hours (A), 8 hours (C), and 24 hours (E) after preparation. (B, D, F) Frequency histogram of DLS measurements of the mean hydrodynamic diameter (nm) found in CCD suspensions 2 hours (B), 8 hours (D), and 24 hours (F) after preparation. (G) Transfection efficiency measured as the % of GFP-positive cells analyzed by flow cytometry of HEK293T cells seeded in p6-well plates and transfected with CCDs 30 minutes, 2 hours, 8 hours, and 24 hours after preparation. CCDs 30 minutes after preparation (light blue) and PEI at a mass ratio of 3:1 with DNA (orange) are shown as transfection reference values ​​(see Figure 4).

[0040] Figure 7. (A) Relative cell viability after 24 h of incubation with CDs at 0.05 g / L, CCDs at a CD : PEI mass ratio of 20:3 and PEI at 0.016 g / L using the MTT assay. (n=9, mean values ​​± SD. Student’s t test was applied for statistical significance: * P<0.05 *** P<0.001.) (B) Agarose gel electrophoresis of the complexes at different mass ratios.

[0041] Figure 8. (A) Transfection efficiency measured as the % of GFP-positive cells analyzed by flow cytometry of HEK293T cells seeded in 100 mm diameter dishes and transfected with CCDs generated by incubating CDs with PEI at a mass ratio of 20:3 and mixed with a fixed amount of DNA to obtain the optimized mass ratio of 20:3:1. Standard transfection using PEI at a mass ratio of 3:1 with DNA was used as a control (orange) (n=11, mean values ​​± SD). Student’s t-test was applied for statistical significance: ***P<0.001). (B) Viral transduction efficiency measured as the % of GFP-positive cells analyzed by flow cytometry of HEK293T cells after incubation with viral supernatant secreted by HEK293T cells after co-transfection of three vectors using CCD or PEI conditions (n=9, mean values ​​± SD).(C) Representative fluorescence microscopy images of HEK293T cells virally transduced as in (B) (scale bar = 100 pm). (D) Viral transduction efficiency measured as the % of GFP-positive cells analyzed by flow cytometry of MenSC cells after incubation with viral supernatant secreted by HEK293T cells following co-transfection of three vectors using CCDs or PEI conditions (n ​​= 3, mean values ​​± SD). Student’s t-test was applied for statistical significance: *** P < 0.001). (E) Representative fluorescence microscopy images of MenSC cells virally transduced as in (D) (scale bar = 100 pm).

[0042] Figure 9. Histograms of flow cytometry analysis of HEK293T (A) and MenSC (B) after transduction with viral supernatant produced using transfection under CCDs or PEI conditions. The side panel shows the percentage of GFP-positive cells, as well as the mean and median fluorescence intensity values ​​(related to Figure 8).

[0043] EXAMPLES OF EMBODIMENT OF THE INVENTION

[0044] The present invention is illustrated by the embodiment examples presented below without the intention of limiting its scope of protection.

[0045] Here, we present a simple and affordable method for efficient cell transfection by electrostatically coupling citric acid-based CDs prepared via a hydrothermal method with 25 kDa linear PEI using room temperature stirring. A biofunctional approach is demonstrated to estimate the optimal CD:PEI ratio that can be easily modified and scaled up for specific transfection conditions. The CCDs efficiently bound DNA in nanoparticles, increased cell viability compared to the standard PEI method, and allowed functional expression of exogenous genes, thus avoiding cell degradation. Cell transfection with CCDs increases more than fourfold compared to standard PEI polyplexes, reaching more than 90% transfection efficiency in HEK293T cells.The HEK293T cell line is a reference platform for the challenging large-scale production of virus-based products, requiring the most efficient transfection methods to achieve retroviral generation. The CCDs described above enable the successful production of retroviral particles encoding desired transgenes, confirming their suitability as a transfection method for biological applications and the manufacture of scalable recombinant products.

[0046] Materials and methods

[0047] Materials

[0048] Citric acid (>99.5%) was purchased from Thermo Fisher (Kandel, Germany), and Thermof PEHA (P98%), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), phosphate-buffered saline (PBS), and Triton-X-100 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dialysis tubing with a MWCO of 100–500 Da was obtained from Spectrum Labs, Inc. (Rancho Dominguez, CA). Milli-Q water (18.2 MQ, filtered with a filter pore size of 0.22 pM) was obtained from Millipore and used as the solvent in CD preparation. Dimethyl sulfoxide (DMSO) was supplied by Acros Organics BBVA (Verona, Italy). Polyethyleneimine, linear, MW 25000, transfection grade (PEI 25K™) was obtained from Polysciences Inc (Cat#23966) and a 1 mg / mL solution was prepared in Milli-Q water and equilibrated to pH 7. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), L-glutamine, and penicillin / streptomycin solution were obtained from Gibco.All chemicals were used without further treatment. The bicistronic retroviral vector pMX-GFP-Sox15 has been previously constructed in the laboratory of the inventors of the present invention [8, 9; Figure 1], and plasmids encoding packaging proteins pCMV-GAG-POL and the plasmid encoding envelope protein pCMV-VSV and 6050 bp plasmids were obtained from Addgene repositories (Addgene Cat#14887 and Addgene Cat#8454).

[0049] Carbon dot synthesis

[0050] Citric acid was used as a precursor to generate CDs using a hydrothermal approach. First, an aqueous solution of citric acid (50 g / L) was transferred to a Teflon-lined vessel and placed inside a steel reactor. It was then heated in an oven to 200 °C and left for 4 hours. The vessel was then allowed to cool to room temperature (25 °C) before the reaction products were centrifuged to remove any insoluble material. The solution was then dialyzed for 24 hours to clean the carbon nanoparticles (CDs).

[0051] Optical characterization of CDs

[0052] UV / vis absorption spectra of CDs were obtained using a Jasco V-730 UV-Vis between 200-600 nm. A Photon Technology International (PTI) Inc. QuantaMaster40 spectrofluorometer equipped with a 75 W continuous xenon arc lamp was used. An ASOC-10 USB interface with FeliX GX software was used for fluorescence data acquisition, and the hardware was controlled for all system configurations. The slit widths for excitation and emission were both 2 nm. All optical measurements were performed in a 10 mm quartz cell at room temperature (25°C).

[0053] Dynamic Light Scattering (DLS) An aqueous suspension with a CD or CCD concentration of 50 mg-mL-1 was prepared to obtain size distribution information and zeta potential measurements using dynamic light scattering (DLS, Malvern Zetasizer Nano-ZS90). Measurements were performed in a low-volume disposable cuvette type ZEN0118, setting 2420 as the refractive index with 173° backscatter (NIBS default) as the detection angle. The measurement duration was set to automatic, and three were set as the number of measurements. A general-purpose analysis model (normal resolution) was chosen.

[0054] Transmission electron microscopy

[0055] Transmission electron microscopy (FEI Tecnai G2 Twin microscope) operating at an accelerating voltage of 100 kV was used to evaluate particle shape and size. A drop of an aqueous suspension containing the CDs and CCDs was placed on a carbon-coated copper grid, and the water was allowed to evaporate for TEM analysis.

[0056] Aoarose ge / retardation test

[0057] Agarose gel electrophoresis was carried out to study the binding of CCDs with DNA. Briefly, aqueous solutions of CCDs prepared at a ratio of 20:3 (ug CD:ug PEI) were incubated at different mass ratios of CCDs (100:1, 40:1, 20:1, 10:1) with DNA. The obtained solutions were incubated at room temperature for 30 minutes, followed by agarose gel electrophoresis analysis in TAE buffer for 60 minutes at 90 V. The results were observed using BioRad-image Lab software under UV light. The above-mentioned DNA solutions and PELADN (mass ratio 3:1) were applied as controls.

[0058] Cell line culture conditions, cell transfection and transduction

[0059] The HEK293T cell line was obtained from Sigma Aldñdch (#12022001). Primary somatic MenSC (also called MnSC) cell lines were previously generated after informed consent from the donor and with authorizations from the bioethics committee and the National Research Ethics Service Review Board (#PR-03-2018) [8]. HEK293T cells were cultured with high glucose in DMEM (with 10% FBS, 1% glutamine and 1% penicillin-streptomycin). MenSC cells were cultured in DMEM-F12 containing 10% FBS, 1x NEAA, 1x L-glutamine, penicillin and streptomycin as previously described [8, 9]. Standard transient transfection was followed using either the PEI method (as previously described [9]) or generated CCDs (as described below in the section Effect of CDs on cell viability). For retrovirus production, HEK293T cells were seeded at 70% cell confluence in 100 mm dishes.After 24 hours, cells were transfected with a transgene encoding the pMX viral vector, the Gag-Pol vector, and the VSV-G plasmid using either the polyethyleneimine method or generated CCD particles. The supernatant was collected 24 hours post-transfection and filtered through 45 mm pore size filters. Cell transduction was performed in HEK293T or MenSC cells using 2 mL of unconcentrated viral supernatant to transduce 100,000 cells in the presence of 4 pg / mL of polybrene.

[0060] Cell viability assay

[0061] Cell viability was assessed in HEK293T cells using the MTT assay. Briefly, cells were seeded at a density of 1 x 10 4cells / well in a 96-well plate at 37 °C in a 5% CO2 atmosphere (200 pL per well, number of replicates = 5). After 24 h of culture, the cell culture medium was replaced with fresh medium containing CCDs (or PEI) at different concentrations. After 24 h, the supernatant was replaced with 200 pL / well of fresh medium containing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (0.5 mg-mL-1 ). After 2 h of incubation at 37 °C and 5% CO2, the medium was removed, the formazan crystals were solubilized with 200 pL of DMSO, and the solution was mixed vigorously to dissolve the reacted dye. The absorbance of each well [Abs] was read in a microplate reader (Dynatech MR7000 Instruments) at 550 nm.The relative cell viability (%) and its error relative to the negative control wells containing cell culture medium without nanoparticles and the positive control wells where Triton X-100 was added to the cells were calculated using the following equations:

[0062] RCV(%) = (([Absjtest - [Abs] CtrlPos.) / ([Abs]Neg. Ctrl - [Abs]CtrlPos.)) x100

[0063] Error (%) = RCVtest x SQRT [ (or test / [Abs]test ) 2 + (or test / [Abs]test ) 2 ] where o is the standard deviation.

[0064] Flow cytometry HEK293T cells were harvested at 300xg for 5 min, fixed in 4% v / v formaldehyde for 15 min, centrifuged at 500g for 5 min, and finally resuspended in 0.3 mL of PBS. The percentage of GFP-positive cells was assessed using a flow cytometer (Beckman Coulter Gallios). The 488 nm laser was used for GFP measurement on the FITC-A detector. A total of 100,000 events were analyzed in each sample. First, side scatter height (SSC-H) vs forward scatter area (FSC-A) and FSC-A vs forward scatter height (FSC-H) density plots were used to delineate individual cell populations. The GFP-positive population was then evaluated in a fluorescein isothiocyanate (FITC) vs FSC-A density plot and in a GFP-FITC histogram using cells transfected with GagPol and VSV vectors only (without GFP-encoding vector) as a negative control.Data were analyzed using Kaluza Beckman Coulter software (Beckman Coulter).

[0065] Results and discussion

[0066] Synthesis of CDs and characterization of their optical properties

[0067] Carbon-based nanoparticles (CDs) were hydrothermally generated as described in section 2.2. The CDs display a very uniform, spherical morphology, with an average size of less than 5 nm (Figure 2A) and a hydrodynamic diameter of less than 10 nm (Figure 2B). This synthetic method allows the nanoparticles to have the typical graphite structure, as shown in Figure 2A. Layers of sp2-hybridized hexagonal carbon rings form graphite. HR-TEM images show a distinct lattice fringe at 0.24 nm, revealing that the CDs produced from citric acid also have a graphitic origin.

[0068] The optical properties of the CDs were further examined. The UV / vis spectrum of the CDs shows a small band at 332 nm (Figure 2C) that can be mainly attributed to the np* electronic transition of the surface carbonyl groups (C=O). Photoluminescence (PL) spectra are one of the most crucial properties of CDs in biomedical applications, although some applications, such as cell tracking, require fluorescence emission from the CDs. Many cell biology applications for gene delivery use fluorescent reporter genes or fluorescent fusion proteins designed to follow the effects of exogenous gene constructs on cellular function, and therefore, the PL spectra of the CDs should not interfere with the detection of widely used fluorescent reporter genes, such as GFP, YFP, or RFP.

[0069] In this exemplary embodiment, a bicistronic retroviral vector encoding GFP and the oocyte-enriched factor Sox15 was used for cellular reprogramming after overexpression in target cells [8]. Detection of the GFP reporter gene is used to follow cells expressing the reprogramming factor Sox15 and thus the reprogramming process over time. The fluorescence emission spectra of the CDs showed a PL band at 340 nm (Figure 2D), a typical behavior when CDs are obtained from citric acid at 200 °C, thus confirming that it does not interfere with the detection of the GFP emission peak at -510 nm [9].

[0070] Biofunctional approach for the generation of cationic carbon dots (CCDs)

[0071] Effect of CDs on cell viability

[0072] To generate CCDs by electrostatic bonding of PEI, different amounts of CDs were incubated with PEI with shaking for 3 hours at 25 °C.

[0073] A biofunctional approach was used to establish the CD:PEI ratio for the generation of CCDs.

[0074] First, the optimal concentration of CDs for cell survival was established using the MTT assay, and 0.05 g / L allowed the highest cell viability (Figure 3).

[0075] Optimization of CCD generation by electrostatic bonding with PEI.

[0076] Second, previously optimized control parameters for cell transfection via PEI polyplexes were used using a PEI : DNA mass ratio of 3:1 - 7.5 PEI with 2.5 pg of pMX plasmid DNA per mL of final cell medium per well of a 6-well (p6) plate [8, 9] as a standard starting point for optimization of the CD : PEI : DNA ratio.

[0077] The optimal amount of CDs to combine with PEI was calculated using these two parameters and the optimal amount of CDs for cell survival (50 pg / mL) with the μg of PEI required in the control polyplex transfection mentioned above (7.5 pg). Therefore, the initial CD:PEI:μg test mass ratio was set at 20:3.

[0078] Thus, CCDs were prepared by mixing different amounts of CDs with a fixed amount of PEI to obtain different ratios: 10:3, 20:3, 40:3, 100:3 and 200:3.

[0079] After 30 min, the electrostatically generated CCDs were combined with plasmid DNA in the described fixed amount (2.5 pg per well p6) to maintain the PEI:DNA mass ratio at 3:1, vortexed briefly, and added to the cell medium to test cell transfection efficiency (Figure 4A).

[0080] The most efficient condition for gene delivery was found to be 20:3:1, increasing more than 4-fold the standard “positive” transfection efficiency of PEI polyplexes (Figure 4) and reaching more than 80% GFP-positive cells as measured by flow cytometry and visualized by fluorescence microscopy.

[0081] To confirm that the maximum transfection efficiency was reached, different initial concentrations of CD showing low cellular toxicity (Figure 3), 100 pg / mL and 25 pg / mL, were used in combination with PEI at different CD:PEI mass ratios: 10:3, 20:3 and 40:3 (Table 1 ), confirming that they show a lower efficiency than the use of 50 pg / mL of CD at the 20:3 CD:PEI mass ratio. Table 1. Transfection efficiency of CCD generated at different CD concentrations and CD:PEI mass ratios using a constant PEI:DNA ratio of 3:1. Transfection efficiency is quantified as the % of GFP-positive cells analyzed by flow cytometry. Also shown are relative cell viability (%RCV) measured by MTT assay as in Figure 7A and transfection yield (product of %RCV of cells and transfection efficiency).

[0082] The combination of different amounts of DNA with the CCD generated at the mass ratio 20:3 CD:PEI (Table 2) was also tested and it was confirmed that 1 pg of plasmid DNA (mass ratio 20:3:1 CD:PEI:DNA) was the most efficient condition.

[0083] Table 2. Transfection efficiency of CCD generated at a mass ratio constant of 20:3 CD : PEI using different amounts of DNA. Transfection efficiency is quantified as the % of GFP-positive cells analyzed by flow cytometry.

[0084] The efficiency of CCD transfection in primary cells was also confirmed. Primary cell cultures often display low transfection rates, which complicates their applications. Mesenchymal cells can be obtained from different origins and present broad biomedical applications. Of importance, menstrual blood-derived stromal cells (MenSCs) have recently emerged as cells with relevant regenerative, reparative, and protective properties, and their therapeutic potential is being studied using recombinant protein expression among other technologies; however, there are still very limited transfection studies for gene delivery in MenSCs. CCDs were confirmed to significantly increase MenSC cell transfection (Figure 4D and 4E), opening the possibility of testing these particles for gene overexpression in different primary cell cultures.

[0085] Characterization of CCD size

[0086] The surface charge and particle size of DNA complexes are crucial for gene delivery. It is commonly accepted that complexes within the 40–200 nanometer size range can exhibit improved endocytosis. Below 100 nm, nanoparticle size plays a less important role in potential uptake routes, as the geometry of different endocytic pathways can easily accommodate small nanoparticles.

[0087] The size of the CCDs 30 min after preparation was measured by TEM and DLS (Figure 5). While the CDs were visualized as individual dots less than 10 nm in diameter (Figure 2), the generated CCDs aggregated to form structures larger than 50 nm in diameter (Figure 5A) with an average DLS diameter of approximately 100 nm (Figure 5B).

[0088] The size of the CCDs and DLS diameter increased slightly over time, but remained below ~200 nm 2 h and 8 h after CCD preparation (Figures 6A-D) and formed larger aggregates of ~1 pm after 24 h (Figures 6E and 6F). Transfection efficiency was confirmed to be inversely correlated with CCD size (Figure 6G) and 30 min was maintained as the optimal time to use for cell transfection.

[0089] Effect of CCDs on cell viability

[0090] It was confirmed that the use of CCD improved cell viability compared to PEI and, as expected, slightly decreased cell viability compared to bare CDs, since PEI has been widely shown to affect cell survival (Figure 7A).

[0091] Relative cell viability (%RCV) was evaluated using CCD generated at different CD concentrations and CD:PEI mass ratios (Table 1 ), again showing maximum cell transfection and cell viability with CCD generated at the 20:3 CD:PEI mass ratio shown in Figure 7A. A gel retardation assay was used to confirm the interaction between CCDs and DNA. As shown in Figure 7B, DNA could be completely trapped in the loading slot by both PEI polymers and generated CCDs, whereas bare CDs were unable to bind DNA that migrated along the line, similar to unbound DNA.

[0092] The zeta potential of the complexes was evaluated and found to increase from -0.456 mV in CDs to +18.2 mV in CCDs, confirming their positive charge after electrostatic coating with PEI, allowing efficient interaction with DNA. CCDs present a lower charge than PEI polyplexes (+35 mV), and this effect may explain the lower damage to cell membranes, thus reducing the cytotoxicity observed when using CCDs (Figure 5A).

[0093] Evaluation of CCDs for the generation of retroviral particles

[0094] The protocol for CCD generation described above was used for the challenging generation of retroviral particles by plasmid cotransfection at a different scale to confirm the robustness of the method.

[0095] For efficient retroviral production, 100 mm diameter cell culture dishes are generally used, which have a surface area 6 times larger than that of p6 wells, which involves scaling up the total amount of DNA to 15 pg. In addition, twice the amount of transgene-encoding plasmid is combined with packaging and envelope plasmids in a 2:1:1 molecule:molecule ratio to increase viral production. According to their molecular weight, 7 pg of transgene-encoding plasmid (bicistronic pMX-GFP-Sox15, 6750 bp), 5.7 pg of packaging protein-encoding plasmid (pCMV-GAG-POL, 11 kbp), and 2.3 pg of envelope protein-encoding plasmid (pCMV-VSV, 6050 bp) were used.The optimized CCD generation method described above was then followed, maintaining the efficient CD:PELADN ratio at 20:3:1, thus combining 45 pg of PEI with 300 pg of CDs to generate CCDs which were then mixed with DNA prior to cell transfection into HEK293T packaging cells.

[0096] It was confirmed that CCDs significantly increased cell transfection efficiency after assay scale-up, as shown by flow cytometry quantification of GFP-positive cells (Figure 8A). The adequate production of retroviral particles was then analyzed. For this, cell culture supernatant was recovered 24 hours post-transfection and used to transduce cells. Retroviral particles containing the transgene construct are capable of infecting target cells, integrating into their genome, and expressing exogenous GFP and Sox15 genes.Viral supernatant from CCD transfection showed the highest transduction efficiency in both HEK293T (Figures 8B and 8C) and MenSCs (Figures 8D and 8E), measured as the % of GFP-positive cells (Figures 8B and 8D) and as the level of fluorescence intensity measured by flow cytometry (Figure 9), indicating the highest production of viral particles after CCD transfection.

[0097] References

[0098] 1. Lin-jun Yue, Y.-yW, Jiang-bo Fan, Lin Chen, Qiang Li, Jing-lei Du, Shi-ping Yu, Yong-zhen Yang, Research progress in the use of cationic carbon dots for the integration of cancer diagnosis with gene treatment. New Carbon Materials, 2021. 36(2): p. 373-389.

[0099] 2. Mansuriya, BD and Z. Altintas, Carbon Dots: Classification, Properties, Synthesis, Characterization, and Applications in Health Care-An Updated Review (2018-2021). Nanomaterials (Basel), 2021. 11 (10).

[0100] 3. Yue Liu, J.H., Yan Li, Hai-Ping Wei, Xiao-Shuang Li, Xi-Hua Zhang, Shi-Min Chen, Xiao-Qin Chen, Synthesis of polyethyleneimine capped carbon dots for preconcentration and slurry sampling analysis of trace chromium in environmental water samples. Taianta, 2015. 134: p. 16-23.

[0101] 4. Kong , T. , et al . , -AS 1411 aptamer modified carbon dots via polyethylenimine-assisted strategy for efficient targeted cancer cell imaging. Cell Prolif, 2020. 53(1 ): p. e12713.

[0102] 5. Biswal, M.R. and S. Bhatia, Carbon Dot Nanoparticles: Exploring the Potential Use for Gene Delivery in Ophthalmic Diseases. Nanomaterials (Basel), 2021. 11 (4).

[0103] 6. Gancheng Zuo, A.X., Xihao Pan, Ting Su, Junjian Li, and Wei Dong, Fluorine-Doped Cationic Carbon Dots for Efficient Gene Delivery. ACS Applied Nano Materials, 2018. 1 (5): p. 2376-2385.

[0104] 7. Wang, L., et al., Carbon-dot-based two-photon visible nanocarriers for safe and highly efficient delivery of siRNA and DNA. Adv Healthc Mater, 2014. 3(8): p. 1203- 1209.

[0105] 8. Lopez-Caraballo, L., et al., Analysis of Menstrual Blood Stromal Cells Reveals SOX15 Triggers Oocyte-Based Human Cell Reprogramming. ¡Science, 2020. 23(8): p. 101376. Sanzhez-Mata, A., A. Ferez-Gomez, and E. Gonzalez-Munoz, Protocol to Reprogram Human Menstrual Blood-Derived Stromal Cells to Generate AOX15- IPSCs. STAR Protoc, 2020. 1(3): p. 100183.

Claims

CLAIMS 1. Method for the preparation of cationic carbon dots (CCDs) characterized in that it comprises the incubation of (1) CDs obtained from the hydrothermal treatment of an aqueous solution of amine-free citric acid with (2) PEI (3) using stirring.

2. Method for the preparation of CCDs according to claim 1, characterized in that the CDs used for incubation with PEI have a spherical morphology, with an average size of less than 5 nm in diameter and a hydrodynamic diameter (DLS) of less than 10 nm.

3. Method for the preparation of CCDs according to any of the preceding claims, characterized in that the CDs used for incubation with PEI are CDs obtained from the hydrothermal treatment of an aqueous solution of citric acid.

4. Method for the preparation of CCDs according to any of the preceding claims, characterized in that the PEI used for incubation with the CDs is 25 kDa linear PEI.

5. Method for the preparation of CCDs according to any of the preceding claims, characterized in that the incubation with shaking is carried out for 3 hours at room temperature (25 °C).

6. Method for the preparation of CCDs according to any of the preceding claims, characterized in that the CDs for incubation with PEI are added as an aqueous solution with a concentration of CDs ranging from 0.5 g / L to 0.025 g / L.

7. Method for the preparation of CCDs according to claim 6, characterized in that the CDs for incubation with PEI are added as an aqueous solution with a concentration of CDs ranging from 0.1 g / L to 0.025 g / L.

8. Method for the preparation of CCDs according to claim 7, characterized in that the PEI to be incubated with the CDs is added, obtaining a mass ratio of CD (pg): PEI (pg) that varies from 10:3 to 40:

3.

9. Method for the preparation of CCDs according to claim 8, characterized in that the PEI to be incubated with the CDs is added, obtaining a CD mass ratio (pg): PEI (pg) of 20:

3.

10. Method for the preparation of CCDs according to any of the preceding claims 7 to 9, characterized in that the CDs for incubation with PEI are added as an aqueous solution with a CD concentration of 0.05 g / L.

11. CCDs resulting from a preparation method according to any of the previous claims.

12. CCDs according to claim 11 characterized in that they have a spherical morphology, with an average size greater than 50 nm in diameter and an average hydrodynamic diameter of approximately 100 nm, 30 minutes after preparation of the CCDs.

13. Gene delivery system comprising DNA combined with CCDs according to any of the preceding claims 11 or 12.

14. Gene delivery system according to claim 13, characterized in that it has a mass ratio CD (pg) : PEI(pg) : DNA(pg) that varies from 20:3:0.2 to 20:3:

5.

15. Gene delivery system according to claim 14, characterized in that it has a mass ratio CD (pg) : PEI(pg) : DNA(pg) that varies from 20:3:0.5 to 20:3:

5.

16. Gene delivery system according to claim 15, characterized in that it has a CD (pg) : PEI(pg) : DNA(pg) mass ratio of 20:3:

1.

17. Gene delivery method comprising incubating the cells to be genetically modified with a gene delivery system according to any of the preceding claims 13 to 16.

18. Gene transfection system for producing retroviral vectors comprising a gene delivery system according to any of the preceding claims 13 to 16, characterized in that the DNA includes a plasmid encoding the transgene in combination with a packaging plasmid and an envelope plasmid.

19. Gene transfection system for producing retroviral vectors according to claim 18, characterized in that twice the amount of plasmid encoding the transgene is combined with the packaging and envelope plasmids, resulting in a molecule:molecule ratio of 2:1:

1.

20. Gene transfection system for producing retroviral vectors according to claim 19, characterized in that the bicistronic plasmid pMX-GFP-Sox15 is used as the plasmid encoding the transgene, the plasmid pCMV-GAG-POL as the plasmid encoding the packaging proteins, and the plasmid pCMV-VSV as the plasmid encoding the envelope proteins.

21. Gene transfection method comprising a gene transfection system according to any of the preceding claims 18 to 20.

22. Gene transfection method according to claim 21, characterized in that the HEK293T cells are (1) cultured with high glucose in DMEM (with 10% FBS, 1% glutamine and 1% penicillin-streptomycin); (2) seeded at 70% cell confluence; and (3) are transfected with a gene transfection system according to any of the preceding claims 18 to 20; 24 hours after transfection, the supernatant is collected and filtered.

23. Gene transfection method according to claim 21, characterized in that the primary somatic cell lines of MenSC stromal cells (also called MnSC) are (1) cultured in DMEM-F12 containing 10% FBS, IxNEAA, 1xL-Glutamine, penicillin and streptomycin; (2) seeded at 70% cell confluence; and (3) transfected with a gene transfection system according to any of the preceding claims 18 to 20; 24 hours after transfection, the supernatant is collected and filtered.

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