Calcium-based nanomaterials and their interrelation with chitosan: optimization for pCRISPR delivery.

There have been numerous advancements in the early diagnosis, detection, and treatment of genetic diseases. In this regard, CRISPR technology is promising to treat some types of genetic issues. In this study, the relationship between calcium (due to its considerable physicochemical properties) and chitosan (as a natural linear polysaccharide) was investigated and optimized for pCRISPR delivery. To achieve this, different forms of calcium, such as calcium nanoparticles (CaNPs), calcium phosphate (CaP), a binary blend of calcium and chitosan including CaNPs/Chitosan and CaP/Chitosan, as well as their tertiary blend including CaNPs-CaP/Chitosan, were prepared via both routine and green procedures using Salvia hispanica to reduce toxicity and increase nanoparticle stability (with a yield of 85%). Such materials were also applied to the human embryonic kidney (HEK-293) cell line for pCRISPR delivery. The results were optimized using different characterization techniques demonstrating acceptable binding with DNA (for both CaNPs/Chitosan and CaNPs-CaP/Chitosan) significantly enhancing green fluorescent protein (EGFP) (about 25% for CaP/Chitosan and more than 14% for CaNPs-CaP/Chitosan). Supplementary Information: The online version contains supplementary material available at 10.1007/s40097-021-00446-1. © Islamic Azad University 2021.

Introduction

Recently, gene editing and therapy have been considered a strategic and highly effective therapy for several genetic disorders and inherited diseases [1-4]. In this manner, smart gene delivery systems must have several features—including acceptable biocompatibility, low cellular toxicity, and considerable transfection efficiency—for their transition from the laboratory to the clinics [5-9]. The technology known as CRISPR/Cas [clustered regularly as interspaced short palindromic repeats/CRISPR-associated protein (Cas)] can provide critical changes in the genome in a very precise manner. This technology is considered promising and highly achievable for developing a one-time cure for inherited diseases [10-14].

Generally, gene delivery systems are based on viral and non-viral vectors [15, 16]. Recent publications have revealed significant transfection efficiency and low cellular toxicity for viral vectors [17, 18]. However, based on immunology, there are several concerns regarding the immune response and other related issues when using viral vectors that may not present any defects for generations but can eventually cause structural problems in human molecular and cellular physiology [19-21]. From another perspective, viral vectors are very expensive, contradicting the mission of medical science and biotechnology to provide advanced techniques, facilities, and materials at minimal cost. Therefore, these viral vectors are not acceptable and cannot enter mass production [22-24].

In contrast, non-viral nano-vectors have gain the attentions of numerous scientists because of their low cost of synthesis and preparation, as well as numerous optimization capabilities, enabling scientists to prepare these vectors for prevention, early diagnosis, and even treatment (in different phases) of various diseases [25-30]. The term "efficacy-toxicity" presents a significant paradox in gene delivery systems. More precisely, considerable efficiency in the gene transfections usually increases the cytotoxicity, while low efficiency in the transfections deduces minimal cellular toxicity, acting as a serious obstacle for using non-viral gene delivery vectors. For these reasons, the synthesis and preparation of low-cost non-viral vectors with low cytotoxicity are of major importance [31].

A wide range of polymers have been researched for the transfection performance of and optimization process for gene delivery vehicles, consuming significant time and money. These polymers are often selected based on their molecular weight (MW), size, zeta potential, and other physicochemical parameters [32-34]. Until now, polyethylenimine (PEI) has been known as the gold standard among non-viral gene delivery vectors, and several studies revealed that the above-mentioned paradox is concerning for this polymer. In general, high MW PEI leads to higher transfection efficiency, but meanwhile demonstrates considerable cytotoxicity hindering its widespread use [35, 36]. Scientists have been trying to use PEI in gene delivery, as some research groups work on its auto-fluorescence and nano to microstructure with low transfection efficiency. Attempts have been made to functionalize these structures with PEI with the hope of reducing its concentration and, therefore, reducing its cytotoxicity; however, still no significant results have been obtained to date [37-39]. Similarly, other scholars have focused on different polymeric materials—including poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA) (with an IC50 value of around 4 µg/mL for the polyplex based on PDMAEMA) [40], polyamidoamine (PAMAM) (with an IC50 value of more than 31 µg/mL for the polyplex based on PAMAM) [41, 42] and poly(l-lysine) (PLL) (with an IC50 value of more than 40 µg/mL for the polyplex based on PLL) [43, 44]—finding similar results in terms of molecular weight and cytotoxicity as well. These results have led to the use of high MW polymers to improve transfection efficiency, yet all suffer from enhanced cytotoxicity. Therefore, the use of polymers alone for gene delivery systems has been questioned.

In recent studies, it has been shown that instead of using compounds with a single functional group, it is better to use highly-branched and complex structures with different functional groups for gene delivery systems [45-49]. Highly-branched structures can crosslink different biological molecules through their three-dimensional (3D) structure, and their multiple terminal groups increase the chance of physical and chemical interactions with different molecules or biological compounds. In this manner, highly-branched poly (β-amino esters) (HPAEs) have shown promising results compared to PDMAEMA, PEI, and PLL [50-52]. Of course, from the perspective of solving the ultimate goal, studies on these highly branched polymers (almost all of those cited previously) were not properly organized and departed from the original objective of developing cost-effective polymer synthesis methods, ease of polymer modification and optimization, as well as material accessibility for different types of patients [3, 53, 54].

In the literature, some reports have used natural cationic polysaccharides containing glucosamine units, chitosan, for gene delivery systems showing interesting results [55-57]. Generally, this unacetylated derivative polysaccharide can form stable complexes in the form of polyelectrolytes with DNA. In addition, chitosan at sizes smaller than 90 nm can form a homogenous complex and condense DNA very effectively [58-60]. Along this line, different nanoparticles have been investigated for various medical applications and certainly for gene transfection efficiency [61-63]. Among them, divalent metal cations including Mg2+, Ca2+, Ba2+, and Mn2+ are able to form meta-stable ionic complexes with DNA through helical phosphates [64-66]. In addition, some studies revealed that calcium phosphate (CaP) could form stable complexes with the backbone of the nucleic acids and impart some stabilizing functions to the specified DNA structure [67, 68]. In the next stage, the stable complexes are carried through ion channels across cell membranes to mediate endocytosis, which leads to DNA intracellular distribution internalized via CaP. However, the gene transfection efficiency of these carriers was at very low percentages (almost below 15%) due to the rapid degradation of most of the endocytosed DNA and cytosol excreted, as well as a negligible fraction of the remaining exogenous DNA, which is vital for gene transfection and leads to poor transfection efficiency. Although CaP is considered as a simple, cost-effective, and efficient gene delivery system for in vitro transfection, it has limitations for in vivo use, including easy degradation after delivery of the genetic materials [69-71].

Taking all of the above into account, in this study, we investigated both the calcium nanoparticle and calcium phosphate effect on the transfection efficiency of pCRISPR, and further determined the cytotoxicity of these nanoparticles to a model HEK-293 cell line. Additionally, the effect of the green synthesized calcium nanoparticles (CaNPs) (using Salvia hispanica) on gene delivery was evaluated (Fig. 1).

Fig. 1

A schematic illustration of the synthesis of CaNPs, CaP and their combination with chitosan and pCRISPR formation on the surface of the nanomaterials for gene delivery and transfection along with its possible influence on the HEK-293 cell line

Methods

Preparation of CaNPs–chitosan

Briefly, a homogeneous chitosan solution was prepared by adding 50 mg of chitosan powder into 7 mL deionized water. Then the resultant solution was sonicated for 30 min (with the assistance of a probe sonic; 40 kHz). In the next step, 6 mg of the green synthesized CaNPs was added to the solution, and the pH value was adjusted to 11. The resultant suspension was then heated in an oven at 60 °C for 3 h, forming a homogenous hydrogel. The synthesized nanomaterial was exposed to UV irradiation (280 nm) for about half an hour for sterilization before any biological assays.

Preparation of CaP-chitosan

For Ca–P–Chitosan preparation, a homogeneous chitosan solution was prepared by adding 85 mg of the chitosan powder into 11 mL of deionized water (DI), with the final solution sonicated for about 42 min (with the assistance of a sonic probe; 40 kHz). In the next step, 12 mg of the synthesized CaP was added to the solution, and the pH value was adjusted to 11. The prepared suspension was then heated in an oven at 60 °C for 3 h, forming a homogenous hydrogel. The synthesized samples were exposed to UV irradiation for about half an hour for sterilization before any biological assays.

Preparation of CaNPs and CaP-Chitosan

In this step, the protocol followed was similar to the one mentioned above. After the preparation of the homogenous chitosan solution, both CaNPs and CaP were added to the solution at an equivalent molar ratio, and the final pH was adjusted to 11. The prepared suspension was heated in an oven at 60 °C for 3 h, forming a homogenous hydrogel.

Results

FE-SEM observations

Based on the literature, the synthesized CaNPs should have a semi-spherical morphology with partial hexagonal structures [72, 73]. In this study, both of these structures were obtained using a green CaNP synthesis method. However, due to the use of a plant in this synthesis method, the morphology was slightly unclear compared to published articles that did not use plants for synthesis. However, the spherical nature of the nanoparticles was quite clear in this study. Also, a small amount of residue from the plant can cause aggregation [74, 75], as evident in the reported images.

In the present work, the aim was to synthesize nanoparticles and nanomaterials with very low cytotoxicity, and therefore the plant was used. According to recent studies [76, 77], the general morphology of the nanosystem did not have a major impact on the gene delivery process; therefore, we assumed that the green synthesized nanoparticles and nanomaterials in this study were acceptable for gene delivery applications. The FE-SEM of CaP (Fig. 2) showed that the synthesized CaP were spherical with an occasional clustering morphology, which is also an acceptable morphology agreeing with literature [78, 79]. In addition, chitosan has its own structural fingerprint [80-82] and the synthesized nanomaterials containing chitosan showed a clear non-flat and intertwined surface (Fig. 2). After adding chitosan, the FESEM images showed an almost homogenous and fully-covered nanostructure with chitosan, which could help the genetic material and other types of biomolecules increase surface interactions. It should be noted that this type of nature-inspired polymer also helps scientist increase the stability of the non-viral vector for further biomedical applications, even under harsh conditions. All of the discussed results were in good agreement with the TEM images as well (Fig. 3). TEM images demonstrated that in the presence of CaP and Ca NPs nanoparticles, the role of the chitosan substrate decreased and vanished due to the full coverage of these NPs.

Fig. 2

FESEM images of the synthesized nanomaterials

Fig. 3

TEM images of the synthesized nanomaterials; A chitosan, B CaP, C Ca NPs, D CaP Chitosan, E Ca NPs Chitosan, and F CaP/Ca NPs Chitosan. The scale bar is 100 nm

XRD results

The synthesized nanoparticles and nanomaterials were characterized using XRD. In the XRD pattern for the synthesized CaNPs, the indicator planes of (100), (101), (102), (110), (111), (200), (201), (202), and (211), corresponding to the 2 Theta degrees of 28.5, 34, 47, 50.5 and 54.3 for the first five planes, conclude the successful synthesis of the CaNPs [83, 84]. In addition, the XRD pattern of CaP is in good agreement with JCPDS 9-0432, and demonstrated that the water peaks in the FTIR spectra did not cause structural deformation or changes in CaP [85, 86]. Furthermore, it is fair to assume that with the combination of these nanoparticles with chitosan, the peaks became slightly wider as the crystallinity decreased. As clearly shown in the XRD pattern of the synthesized nanomaterials, the indicator diffractions were slightly wider than the pure nanoparticles but the structure of the synthesized nanoparticles remained intact, indicating the successful incorporation of these synthesized nanoparticles into the chitosan structure (Fig. 4A).

Fig. 4

Physicochemical properties of the synthesized nanoparticles and nanomaterials, A powder XRD (PXRD) pattern and B FTIR spectra

FTIR results

The synthesized nanoparticles and nanomaterials were characterized using FTIR. In the Ca NP spectrum, a peak around 870 cm−1 indicated Ca-O-Ca bonding, while another peak at 710 cm−1 is an indicator of Ca-O bonding (Fig. 4B). In addition, a series of broad peaks at around 3500, 2950, 1790, and 1430 cm−1 belong to hydroxyl, carboxylic acid, amine, and amide functional groups on the surface of the green synthesized CaNPs or in the media of the green synthesis protocol, respectively. The CaNP FTIR spectrum is in good agreement with the previous reports as well [87-89].

In the CaP spectrum, two peaks at around 1040 cm−1 and 962 cm−1 indicated the presence of phosphate. A very low band at around 950 cm−1—that could overlap with the intense band of phosphate—was assigned to the vibration of the P-OH groups. In the range of 3100–3300 cm−1, a broad band was assigned to the overlapping hydrogen vibrations of the structural hydroxyl stretching vibrations and physically adsorbed water. Also, a low-intensity band at around 1630 cm−1 was assigned to the bending vibrations of the adsorbed water. A low-intensity band at around 560 cm−1 revealed the presence of trace phosphate ions as well. The FTIR spectrum of CaP was in good agreement with the literature [90, 91].

In the FTIR spectra of CaNPs–Chitosan, CaP–Chitosan, and CaNPs–CaP–Chitosan, all of the indicator bands for CaNPs and CaP as mentioned above were observed, and a broad band at about 3400 cm−1 was assigned to the axial O–H and N–H stretching of chitosan which overlapped the C–H stretching band around 2800 cm−1. The band around 1650 cm−1 indicated the axial C=O stretching of the acetamido groups of chitosan, while the semi-broad band around 1580 cm−1 was attributed to the N–H bonds (angular deformation) of the amino groups of chitosan. In addition, broad bands around 1420–1470 cm−1 could be assigned to the N–H angular deformation coupling as well as the C–N axial stretching, which certainly overlapped and was represented as a single broad band. Finally, the fingerprint of the presence of chitosan in the structures was confirmed by bands around 1100 cm−1 and 900 cm−1, which were attributed to the vibration of glycosidic bonds—the C–O and C–O–C stretching bonds, respectively. The FTIR spectra of all of the synthesized nanomaterials are in good agreement with the previous reports [92, 93].

Zeta potential of the nanocomplexes

The physical and chemical properties—including surface charge, size, and hydrophobicity—should be considered to develop an ideal nanocarrier to effectively deliver a wide range of therapeutics [84, 94, 95]. The zeta potential of the CaNPs/pCRISPR was − 11 and − 37 for the M/pCRISPR (M/C) of 10 and 100, respectively (Fig. 5A). Additionally, the zeta potential of the CaP/pCRISPR nanocomplexes was − 6 and − 17 for WR(M/C) of 10 and 100, respectively. In this case, the zeta potential of the CaNPs–Chitosan/pCRISPR and CaP–Chitosan/pCRISPR nanocomplexes was − 4, and − 2 mV for the WR(M/C) = 10, and these numbers shifted dramatically to + 12 and + 11 mV for the WR(M/C) = 100. The zeta potential of the modified chitosan with both of CaNPs and CaP and pCRISPR were + 2, + 5, + 11, + 21 and + 27 for the WR(M/C) = 10, 20, 30, 50 and 100, respectively.

Fig. 5

The results of A zeta potential and particle size, B gel electrophoresis. The first col. is the control, and by shifting to the left, the weight ratio of the nanocarrier to the genetic materials increased by doubling the amount. The first amount (second col.) is WR of 1), and C relative cell viability of the synthesized nanoplexes. Gel electrophoresis, zeta potential, size, and relative cell viability are presented for 72 h as the mean (± SD) from three experiments. (*P-value < 0.05, **P-value < 0.01 and ***P-value < 0.001)

Formation of the nanoplatform

The results of gel electrophoresis (Fig. 5B) showed different interaction strengths between the nanomaterial and pCRISPR with enhancing ratios are demonstrated through the intensity of the pCRISPR migration bonds. Based on the previous work [96], CaP has a weak ability to condense pDNA. Yet, in this study, the synthesized CaP showed better results than what would come from just the synthesis procedure. The same results were found for CaNPs, but interestingly, as the zeta potential of these nanoparticles was negative, gel electrophoresis showed a better outcome resulting from the green synthesis medium of these nanoparticles, which is likely due to the presence of calcium in ionic form and the successful electrostatic interaction with DNA.

Furthermore, the influence of chitosan on the performance of CaP and CaNPs in gene delivery was investigated, as shown in Fig. 5A. By increasing the zeta potential, the slope increased and the genetic material bonding improved due to the higher positive zeta potential.

Cell viability assays

In this part of the study, the cellular toxicity of the synthesized nanomaterials on the HEK-293 cell lines were investigated (Fig. 5C). It was found that all of the synthesized nanomaterials showed considerably high relative cell viability on the HEK-293 cells (at a viability of more than 85%) at different concentrations of the synthesis procedure. PEI and related conjugates (with an IC50 value of more than 25 µg/mL), the presently synthesized nanomaterials and nanoparticles—especially CaNPs–Chitosan (with an IC50 value of about 21 µg/mL), CaP–Chitosan (with an IC50 value of about 16 µg/mL) and CaNPs–CaP–Chitosan (with an IC50 value below 15 µg/mL)—have very low toxicity. This result makes such nanomaterials promising candidates as the next-generation gold standard non-viral gene delivery vector.

In vitro gene expression efficiency

In order to evaluate gene expression efficiency, pCRISPR expressing GFP studies were conducted using the HEK-293 cell line. Comprehensive fluorescence microscopy images were obtained to evaluate EGFP expression on the mentioned cells at different concentrations of the nanomaterials and pCRISPR (Figs. 6A–J and 7) along with optical microscopy (Fig. 6L–O). The results indicate that by enhancing the weight ratio, the gene transfection efficiency significantly increased (Fig. 6K). Additionally, the best result from the gene expression of EGFP was about 25% for the HEK-293 cells for CaP-Chitosan. Following papers in the literature, several factors are associated with transfection efficiency, including the size of the genetic material as well as the polymeric length [97-99] but to the best of our knowledge, so far, no reports have claimed significant transfection efficiency with low cost using an environmentally friendly non-viral vector without costly and difficult optimization. Furthermore, after full consideration of the in vitro studies, CaNPs Chitosan, CaP Chitosan, and CaNPs CaP Chitosan were regenerated and studied under microscopic conditions (Supporting Information; Figs. S1, S2, and S3) showing no significant morphological changes after gene transfection.

Fig. 6

The results from 2D fluorescence microscopy of CaNPs with the WR of 50 (A) and 100 (B); CaP with the WR of 50 (C) and 100 (D); CaNPs Chitosan with the WR of 50 (E) and 100 (F); CaP Chitosan with the WR of 50 (G) and 100 (H); CaNPs/CaP Chitosan with the WR of 50 (I) and 100 (J) (The scale bar is 50 µm); K GFP positive cells for the synthesis materials at different WRs on the HEK-293 cell line; and also optical microscopy of CaP Chitosan with the WR of 20 (L) and 50 (N); and CaNPs/CaP Chitosan with the WR of 20 (M) and 50 (O) (magnification; × 80). 2D fluorescence microscopy and EGFP are presented as the mean (± SD) from three experiments. (*P-value < 0.05, ***P-value < 0.01 and ****P-value < 0.0001)

Fig. 7

2D fluorescence microscopy results of the CaNPs Chitosan (A–D) and CaP Chitosan (E–H) on the HEK-293 cell line. The scale bar is 10 µm

Insight into the mechanism

Investigating the effect of different types of nanoparticles and nanomaterials as non-viral gene delivery vectors on different cell lines is of great importance. The ability of endosomal escape is one of the most important parts for the biomedical applications, especially in case of gene therapy and delivery. In this regard, the underlying mechanism for this effect has been called a proton sponge. The proton sponge effect is the ability of basic molecules to be protonated resulting in endosome bursting. In this study, one of the main reasons for the great efficiency of these synthesized nanomaterials is their buffering capacity. To evaluate their buffering capacity, adsorption ability studies of the nanostructures in the presence of different transition metals have been performed. The sorption isotherms of Cd2+, Cu2+, and Pb2+ of the synthesized nanoparticles and nanomaterials were demonstrated the H and L types of Giles classifications [100, 101] (Fig. 8).

Fig. 8

The sorption isotherms of Cu2+, Pb2+ and Cd2+ on the synthesized CaNPs/Chitosan (A), CaP/Chitosan (B) and CaNPs/CaP/Chitosan (C)

Discussion

Previous works showed that [102, 103] the good nanocarrier for gene delivery procedure should have higher positive zeta potential. In addition, shifting to a negative phase was expected by adding pCRISPR to the CaNPs and CaP nanocomplexes [104, 105]. Furthermore, some papers emphasized successful gene transfection with negative or even neutral zeta potential [106], but based on our knowledge, to get materials into the clinical or industrial phase, results have to be extraordinary; in this manner, a positive zeta potential is critical. This phenomenon, shifting the zeta potential by changing the concentration and weight ratio, could be correlated to the presence of different types of functional groups on the surface, leading to significant aggregation and agglomeration. These aggregations and interactions change the microenvironment around the nanomaterials, which in turn will change zeta potential and particle size.

Physical, chemical and biological stability of a nano-platform for every biomedical application is of great importance [107-109]. Amine functional groups of chitosan can increase the efficiency of interactions with the pCRISPR phosphate backbone, and interestingly, this mechanism is considered in the wide range of pH and physicochemical conditions. From another perspective, it has been revealed that CaP has an ability for DNA condensation; therefore, CaP possesses a synergic effect on pCRISPR condensation along with chitosan. Until now, there have been no reports investigating this synergic effect, including the role of calcium itself. In this study, the ability of CaNPs to condense genetic material was investigated. However, the hydroxyl groups on the surface of the nanoparticles make their zeta potential shift more towards the negative region, and the potential electrostatic interaction between those nanoparticles and pCRISPR are quite weak. But, the ability of the Ca NP synergic effect on CaP and chitosan is still unknown, and this research was conducted to understand such phenomena.

The effect of the presence of both CaP and CaNPs in the chitosan structure revealed very interesting results in terms of both relative cell viability and gel electrophoresis analysis. In this regard, the nanoparticles have a synergic effect on each other in the chitosan structure, and they likely prevent probable aggregation through electrostatic forces and via the functional groups on their surface. Furthermore, another theory can be put forward herein that the calcium nanoparticle can cause charge separation on the surface of the chitosan in the presence of calcium phosphate; thus, it can reduce the number of nanoparticles—or rather, the nanomaterials—that accumulate. These accumulated nanomaterials then reduce the size of the final nanomaterial that condense DNA, which lead to a reduction in the size of the final nanomaterial improving transfection efficiency, both of which are revealed in this study.

Generally, by increasing the ratio to above 2, the migration of pCRISPR was confined, which is proof of genetic material condensation by the synthesized nanomaterials and nanoparticles in this study at low weight ratios. Therefore, the pyrolysis time was exactly selected to be an optimal balance between the surface modification of chitosan and N-doping, leading to significant gene condensing.

Based on the literature [110-112], by increasing the time of radiation, cytotoxicity decreases gradually due to the deterioration of amino groups, which was also exhibited in this research. However, this trend was not observed for the synthesized CaP and CaNPs, due to the absence of nitrogen-based compounds. Additionally, by increasing the PEI concentration on the surface, the relative cell viability reduced with much higher slope compared to the synthesized CaNPs–Chitosan, CaP–Chitosan, and CaNPs–CaP–Chitosan [76, 113]. This can be correlated to the chemistry of chitosan and the synergic effect of those nanoparticles in condensing pCRISPR and preventing aggregation. In comparison with PEI, this makes amino groups less accessible, and, as a result, an increase in the concentration of the synthesized nanomaterials reduces cell viability rate, resulting in a less steep slope. All in all, these results are consistent with the literature [114, 115]. Our findings are considered the first to report on replacing PEI with a semi-green and cost-effective, yet more effective, nanomaterial in pCRISPR delivery [116, 117].

In this work, the used metal ions can affect the environmental pH, and the synthesized nanomaterial buffering capacity inhibits the unwanted effect of other parameters on changing pH values. The pH of the nanocomplexes on the sorption process of the metal ions was recorded at 7.4 to 8.6, which could be considered as proof that these metals were in metal hydroxide form [118, 119].

This work aimed to investigate the role of carbon-based nanomaterials and different forms of calcium in green media as smart gene delivery systems. In this case, CaNPs and CaP were selected as different forms of calcium, and chitosan was selected as the carbon-based carrier. Different nanomaterials were prepared based on the above-mentioned calcium forms and chitosan, and were fully characterized in terms of their chemical, physical and biological properties. The chemical characterization proved the successful synthesis of these nanoparticles and nanomaterials, and fully biomedical characterization represents a significant ability of the genetic material to make a bind to the nanoplatform, with having considerably high relative cell viability.

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 503 kb)