Characterization of gene-loaded nanoparticles

Gene-loaded nanoparticles were developed based on the self-assembly properties of dendritic macromolecules and genes. Subsequently, a series of experiments were conducted to characterize the morphology, diameter, and charge of the nanoparticles. Finally, it was verified that the nanoparticles were successfully loaded with the gene of interest by analyzing various chemical elements contained in dendritic macromolecules and genes.

Branched polyethylenimine (bPEI) is a recent discovery in cationic polymer gene transfection technology. bPEI has been utilized in a wide range of hosts and is characterized by its ease of use, low cytotoxicity, and high transfection efficiency.21–2321. S. Yang, X. Zhou, R. Li, X. Fu, and P. Sun, Curr. Protoc. Chem. Biol. 9(3), 147–157 (2017). https://doi.org/10.1002/cpch.2522. P. A. Longo, J. M. Kavran, M. S. Kim, and D. J. Leahy, Methods Enzymol. 529, 227–240 (2013). https://doi.org/10.1016/B978-0-12-418687-3.00018-523. L. Xue, Y. Yan, P. Kos, X. Chen, and D. J. Siegwart, Drug Delivery Transl. Res. 11(1), 255–260 (2021). https://doi.org/10.1007/s13346-020-00790-9 Herein, a biguanide-modified-4-aminobenzoic acid (BGBA)-modified bPEI (BGBA–bPEI) was designed, which could readily adsorb plasmids and siRNAs electrostatically and self-assemble to form nanoparticles, thus encapsulating genes within the nanoparticles. BGBA–bPEI is a large positively charged molecule that is uniformly dispersed in water (Fig. S1). When a negatively charged gene is added to the solution, the BGBA–bPEI molecule and the gene are attracted to each other as a result of their dissimilar charges. Finally, nanoparticles loaded with the gene of interest were obtained [Fig. 1(a)]. Based on the principle presented in the schematic diagram, nanoparticles comprising BGBA–bPEI, with siRNA as an example, were prepared and their morphology and particle size were observed using transmission electron microscopy (TEM). The resulting nanoparticle size was approximately 200 nm. The size was relatively uniform and dispersed in a liquid state, thus indicating that the process of constructing gene-loaded nanoparticles was successful [Fig. 1(b)].

To stably synthesize circRNA-3503 in the lesion, plasmids that could transcribe circRNA were constructed by purchasing a vector specifically designed for circRNA transcription [Fig. S2(a)]. The sequence of our target gene was integrated into this vector [Fig. S2(b)]. The resulting product was transferred into bacteria and plasmid DNA was isolated and sequenced from individual colonies. The correct clone was defined as having a vector that could successfully transcribe circRNA-3503 [Fig. S2(c)]. To verify that the gene sequence did not form a circle, divergent and convergent primers were designed. Sequences that successfully formed a circle indicated that they were successfully transcribed into RNA [Fig. S3(a)]. Based on our experimental results, groups A3 and B3 exhibited bright bands, indicating that RNA was readily transcribed from both sequences [Fig. S3(b)]. Finally, it was verified that the constructed plasmid could successfully transcribe circRNA-3503.

Different proportions of BGBA–bPEI were mixed with plasmids. Using dynamic light scatterer (DLS), it was revealed that when the mass ratio of BGBA–bPEI and plasmid was 1:1, the distribution of nanoparticles was extremely wide, ranging from small to large nanoparticles, which indicated the poor stability of the composite system. When the mass ratio was increased, the nanoparticles tended to be more stable. Furthermore, when the mass ratio was increased to 10:1, the particle size was approximately 220 nm and the distribution was relatively concentrated. Nanoparticles loaded with siRNAs at a mass ratio of 10:1 were constructed, and the diameter of the resulting nanoparticles was observed to be stable at approximately 230 nm, thus demonstrating that nanoparticles loaded with genes can be successfully produced at a mass ratio of 10:1 [Fig. 1(c)]. In addition, by characterizing the surface potential, it was determined that with an increase in the BGBA–bPEI mass ratio, the surface potential of the nanoparticles gradually tended to be positive [Fig. 1(d)]. Based on the above measurements, when the mass ratio of BGBA–bPEI was relatively low, the diameter of the nanoparticles was 100–200 nm, and even a large percentage of nanoparticles were smaller than 100 nm. However, as the mass ratio increased, the diameter of the nanoparticles was concentrated around 200 nm, indicating that these nanoparticles were in a relatively stable state. Although nanoparticles with a low mass of BGBA–bPEI were small in diameter, they exhibited a negative charge, which is not conducive to uptake by cells. Therefore, nanoparticles with a mass ratio of 10:1, which were more stable, more uniform in diameter, exhibited a positive charge, and more conducive to cell uptake, were used. Next, an energy spectrum analysis of the gene-loaded nanoparticles was performed, and it was found that the interior of blank nanoparticles was rich in C and O elements. As bPEI contains phenyl boric acid, blank nanoparticles also contained the boron element. However, phosphorous (P) elements were extremely limited [Fig. S4(a)]. Subsequently, the nanoparticles loaded with plasmids and siRNA, respectively, were examined, and it was found that in addition to being rich in C, O, and B, these nanoparticles had higher amounts of P than blank nanoparticles. This P element was provided by the genes loaded on the nanoparticles [Fig. S4(b)]. Thus, nanoparticles loaded with our gene of interest were successfully constructed. To further verify that our synthesized nanoparticles were successfully loaded, a mapping energy spectrum of the nanoparticles was generated [Fig. 1(e)]. The inside of the nanoparticle was rich in P, C, and O elements, which indicated that the nanoparticles were successfully loaded.

Characterization of a heat-sensitive nanocomposite hydrogel

The most important characteristic of nanocomposite hydrogels is the thermal response. The appropriate concentration of hydrogel was determined using a phase transition experiment. Subsequently, rheological and macroscopic experiments were conducted to test the properties of the nanocomposite hydrogel transitioning from the liquid to solid state at 37 °C. PLGA (poly(lactic-co-glycolic acid))–PEG (polyethylene glycol)–PLGA self-assembles to form micelles in an aqueous solution, thereby establishing a hydrogel system when combined with water. At room temperature, the micelles were randomly distributed and moved freely, indicating the liquid state of the hydrogel. Upon heat-induced stimulation, the micelles exhibited a regular linear arrangement and formed an interlaced micelle network. Finally, the hydrogel solidified [Fig. 2(a)]. Based on the principle described in the schematic diagram, PLGA–PEG–PLGA triblock copolymers with a D,L-lactide (LA) to glycolide (GA) ratio of 3.6 were prepared (as determined using 1HNMR), and the molecular weight of PLGA–PEG–PLGA was 1740–1500–1740 kDa (Fig. S5). The amphiphilic PLGA–PEG–PLGA triblock polymer self-assembled to form nanomicelles in water, with an average particle size of approximately 40 nm [Fig. 2(b)]. The triblock polymer was tested at various concentrations (25, 22.5, 20, 17.5, and 15 wt. %) to determine the changes in the state of the polymer water system at increased temperature. Figure 2(c) shows that the sol–gel transition temperature of the polymer water system decreased with an increase in polymer concentration. When the polymer concentration was 25 wt. %, the phase transition temperature reached 33 °C and a gel was formed through in vivo injection. We further verified the phase transition of the polymer water system at a concentration of 25 wt. % using rheological experiments and determined its rheological properties after adding the nanocomposite system. The results [Figs. 2(d) and 2(e)] revealed that the addition of the nanocomposite system did not affect the phase transition temperature or modulus of the whole system. To characterize the sol–gel transition of the polymer hydrogel system at a macroscopic level, we acquired a macroscopic image of the system [Fig. 2(f)]. The polymer hydrogel system was transparent and flowed easily in its “sol” state at 25 °C. When the temperature was increased to 37 °C, it transiently transformed into a nonflowing, semisolid gel. After returning to room temperature, the gel gradually returned to a flowing sol state. Subsequently, we mixed the polymer hydrogel [Fig. S6(a)] and nanoparticle system [Fig. S6(b)] according to the designed mass ratio and the nanocomposite hydrogel was prepared [Fig. S6(c)]. The gel–solution transition experiment was repeated. The results indicated that the nanocomposite hydrogel system retained good heat-sensitive properties and underwent solidification during heat stimulation. These findings indicate that the gels formed using this system are reversible and meet the application requirements of various disease models.

Biocompatibility of the nanocomposite hydrogel

The cytotoxicity and biocompatibility of biological materials should always be tested. We measured cell proliferation using the Cell Counting Kit-8 (CCK-8) assay, which indirectly reflects the cytotoxicity of biomaterials. We also used live/dead cell staining technology to stain and visually observe and count the dead cells. The cytotoxicity of the nanoparticles and nanocomposite hydrogels was examined. To mimic the application of the hydrogel, MSCs were selected as experimental cells and various concentrations of the biomaterials in MSC cultures were examined. The concentrations of the nanoparticles ranged from 10 to 1000 μg/ml, whereas the nanocomposite hydrogel ranged from 50 to 1600 μg/ml. After culturing for 48 h, the activity of the MSCs was measured using the CCK-8 assay. Using a series of MSC cultures treated with nanoparticles, the OD values of the experimental and control groups were not significantly different. Therefore, the nanoparticles exhibited no apparent cytotoxicity at the tested concentrations [Fig. S7(a)]. Furthermore, in a series of MSC cultures exposed to nanocomposite hydrogels, the OD values of the experimental and control groups were also similar [Fig. S7(b)]. Therefore, the nanocomposite hydrogels exhibited good biocompatibility and can be safely injected in vivo.

Biological effects of nanocomposite hydrogels for the treatment of ONFH

The nanocomposite hydrogels were designed to regulate Bcl-2 and PPARγ in order to inhibit apoptosis and adipogenic differentiation of stem cells, respectively. Western blot analysis was used to assess the expression of the two target proteins in stem cells following exposure to the nanocomposite hydrogel. Flow cytometry was used to detect apoptosis associated with the nanocomposite hydrogel. The inhibitory effect of the nanocomposite hydrogel on adipogenic differentiation of stem cells was examined using a triglyceride kit.

We examined the underlying mechanism for the effects of the nanocomposite hydrogels for the treatment of ONFH. Our specially designed plasmid was designed to utilize the transcription system of MSCs to transcribe circRNA-3503. This circRNA can theoretically enhance the expression of Bcl-2 and inhibit alcohol-induced apoptosis of MSCs by acting as an RNA sponge and neutralizing micRNA-181c-3p. The siRNA-containing nanoparticles inhibit the expression of PPARγ in MSCs through the release of siRNA, thereby inhibiting the alcohol-induced adipogenic differentiation of MSCs. We confirmed that the normal physiological function of the MSCs was restored [Fig. 3(a)]. We added ethanol (0.09 mol/l) to the medium of the MSCs to simulate the ethanol-containing microenvironment that occurs in patients with ONFH. We used different biomaterials at various concentrations during coculture with the MSCs and verified the mechanism of MSC regulation following exposure to nanocomposite hydrogels. First, to identify the mechanism through which plasmid-loaded nanocomposite hydrogels inhibit MSC apoptosis during exposure to ethanol, we established four groups. Untreated MSCs were used as the control group, MSCs exposed to ethanol were designated the alcohol group, MSCs exposed to ethanol and blank nanocomposite hydrogels were designated the hydrogel group, and MSCs exposed to ethanol and plasmid-loaded nanocomposite hydrogels were designated the [email protected] group. To measure the inhibition of alcohol-induced apoptosis of MSCs by the nanocomposite hydrogels, flow cytometry was used [Fig. 3(b)]. The apoptosis rate of the Alcohol group was significantly higher compared with that of the control group, which indicates that long-term drinking results in apoptosis of human stem cells. However, there was no significant difference in the apoptosis rate of the alcohol-combined hydrogel group, indicating that the influence of irrelevant variables could be excluded. The apoptosis rate of the [email protected] group was significantly lower compared with that of the alcohol group. The plasmid-loaded nanocomposite hydrogel significantly inhibited alcohol-induced apoptosis of the MSCs [Fig. 3(c)]. To verify the mechanism of plasmid-induced inhibition of MSC apoptosis, we assessed the expression of Bcl-2 in the MSCs by western blot analysis [Fig. 3(d)]. Quantitative analysis of the results revealed that under regulation by the plasmid, Bcl-2 levels in the [email protected] group were significantly higher compared with that in the alcohol group [Fig. 3(e)]. Therefore, the nanocomposite hydrogel loaded with plasmids increased Bcl-2 levels in the MSCs, thus inhibiting apoptosis.To verify whether the nanocomposite hydrogels loaded with siRNA could inhibit the alcohol-induced adipogenic differentiation of MSCs, we established four experimental groups. Untreated MSCs were used as a control group, MSCs exposed to alcohol were designated the alcohol group, MSCs exposed to alcohol and blank nanocomposite hydrogel were designated the hydrogel group, and MSCs exposed to alcohol and siRNA-loaded nanocomposite hydrogel were the [email protected] group. PPARγ, a key intracellular protein that promotes the adipogenic differentiation of MSCs, was assessed by western blot analysis [Fig. 3(f)]. Quantitative analysis of the western blot results indicated that the PPARγ levels in the Alcohol group were significantly higher compared with that in the control group, indicating that long-term alcohol consumption results in lipid accumulation in the femoral head, thus aggravating ONFH. No significant differences were observed between the Hydrogel and Alcohol groups. Most importantly, PPARγ levels in the [email protected] group were significantly lower compared with that in the alcohol group, indicating that the nanocomposite hydrogel loaded with siRNA significantly inhibits the alcohol-induced expression of PPARγ in MSCs [Fig. 3(g)]. To further verify whether siRNA-loaded nanocomposite hydrogels inhibit the adipogenic differentiation of MSCs, we measured triglycerides in the MSCs. The triglyceride levels of the alcohol group were significantly higher compared with that in the control group, whereas the triglyceride levels of the [email protected] group were significantly compared with that in the alcohol group [Fig. 3(h)]. Thus, the nanocomposite hydrogel microspheres significantly inhibit the alcohol-induced adipogenic differentiation of MSCs.

Taken together, we designed a nanocomposite hydrogel that inhibits alcohol-induced apoptosis and adipogenic differentiation of MSCs by regulating the expression of Bcl-2 and PPARγ.

Treatment of ONFH using nanocomposite hydrogels in vivo

To further validate the efficacy of the nanocomposite hydrogels for the treatment of alcohol-induced ONFH, animal experiments were performed. The effective duration of nanocomposite hydrogels in the femoral head and its effect on the pathological changes of the femoral head were evaluated. We designed a fluorescence residue assay to detect the time of effect of the nanocomposite hydrogels. The bone status of femoral head was assessed by micro-computed tomography (CT). Tissue sections were prepared and immunohistochemical staining was used to observe apoptosis of cells in the femoral head and the secretion of osteoblast-related proteins. Based on the above data, we evaluated the pathology of ONFH and the progress of repair.

We generated a model of alcoholic ONFH by feeding rats with a commercially available ethanol-containing diet [Fig. 4(a)]. The rat hip joint was injected to treat ONFH [Fig. 4(b)]. The rats were acclimated to the alcohol diet for 10 days. The proportion of the alcohol diet was increased by 20% every two days up to 100% on the 10th day. The control rats were fed a control diet, and the experimental rats were fed the alcohol diet. The daily intake for each rat was limited to 100 ml. An injection needle containing a needle core was used for intra femoral head injection. This prevents the rat tissue from clogging the needle. We inserted the needle approximately 1 mm below the trochanter of the femur. The angle between the needle and the femoral shaft was approximately 120° and the insertion depth was approximately 1 cm. We verified that the needle tip reached the femoral head by x-ray. The needle core was removed and the hydrogel was injected.To verify the degradation rate and retention time of the nanocomposite hydrogels in vivo, we established a fluorescence residue assay. The hydrogel was labeled with a Cy-5.5 fluorescent dye and injected into the hip joint of the rats, and the residual dose of the fluorescent dye was detected every 10 days [Fig. 4(c)]. The results indicated that the fluorescence value in the rats was approximately 55.0% of the initial value on day 20. On day 40, the fluorescence value in the rats was approximately 9.3% of the initial value, indicating that the nanocomposite hydrogel can release nanoparticles in rats for approximately 40 days [Fig. 4(d)]. Therefore, our nanocomposite hydrogel was stable in rats and achieved long-term activity.After treatment of alcoholic ONFH, micro-CT was performed [Fig. 4(e)]. The micro-CT images revealed that the tissue in the medullary cavity of the rat femoral head in the control group was dense and uniform, whereas the alcohol and hydrogel groups exhibited large areas of dark necrotic tissue in the femoral head. Areas of necrotic tissue in the rats by the eighth week were significantly larger compared with that in the fourth week. ONFH was significantly improved in the treated [email protected] group compared the alcohol and hydrogel groups. Subsequently, bone mineral density (BMD) [Fig. 4(f)], bone volume (BV)/trabecular volume (TV) [Fig. 4(g)], and Tb.Th [Fig. 4(h)] were assessed by micro-CT. A statistical analysis revealed that BMD, BV/TV, and Tb.Th of the control group were significantly higher compared with that of the alcohol group in the fourth and eighth weeks. The results indicate that alcohol destroys the bone of the femoral head in rats and induces ONFH. The Tb.Th of the [email protected] group was significantly higher compared with that of the alcohol group in the fourth week. By the eighth week, BMD and BV/TV of the [email protected] group were significantly higher compared with that of the alcohol group. Therefore, following administration of the nanocomposite hydrogel, the bone destruction and necrosis in the femoral head were effectively inhibited. However, no significant differences between the experimental groups were observe in several cases, which may be the result of small fluctuations in physiologically relevant values or an inadequate number of samples. We plan to conduct future experiments by increasing the number of samples.Finally, we tested the effect of the nanocomposite hydrogel on bone repair in the necrotic femoral head area. After treatment, the femoral heads of the rats were harvested for tissue sectioning, Hematoxylin and eosin (H&E) staining, and immunohistochemical staining. We defined the pathological manifestations of alcoholic ONFH as the presence of diffuse granular vacuolar cells in the trabecular bone with pyknotic nuclei and necrosis in the surrounding bone marrow.24–2624. T. Ichiseki, A. Kaneuji, Y. Ueda, S. Nakagawa, T. Mikami, K. Fukui, and T. Matsumoto, Arthritis Rheum. 63(7), 2138–2141 (2011). https://doi.org/10.1002/art.3036525. Y. X. Chen, D. Y. Zhu, J. H. Yin, W. J. Yin, Y. L. Zhang, H. Ding, X. W. Yu, J. Mei, Y. S. Gao, and C. Q. Zhang, Oncotarget 8(59), 100691–100707 (2017). https://doi.org/10.18632/oncotarget.1916026. Y. X. Chen, S. C. Tao, Z. L. Xu, W. J. Yin, Y. L. Zhang, J. H. Yin, Y. S. Gao, and C. Q. Zhang, Oncotarget 8(19), 31065–31078 (2017). https://doi.org/10.18632/oncotarget.16075 H&E staining [Fig. 5(a)] revealed no histopathological changes associated with ONFH in the control group; however, in the alcohol group, diffuse vacuolar areas (black arrows) were observed in the trabecular bone of the femoral head and a large amount of necrotic cell debris had accumulated in the medullary cavity. This indicated that the alcohol-induced ONFH rat model was successfully established. Meanwhile, a significant reduction in osteonecrosis was observed in the [email protected] group, indicating that the nanocomposite hydrogel had a significant effect on ONFH. To further analyze the histopathological results, the vacuoles in the trabecular bone were analyzed [Fig. 5(b)]. The alcohol group had significantly more vacuoles compared with the control group; however, there was no significant difference in the number of vacuoles between the alcohol and hydrogel groups, and thus, irrelevant variables were excluded by comparing the two groups. Most importantly, the number of vacuoles in the [email protected] group was significantly lower compared with that in the alcohol group, indicating that the nanocomposite hydrogel significantly inhibits disease progression.To detect apoptosis in the necrotic tissue of the femoral head, we performed a cleaved caspase-3 immunohistochemical staining of rat femoral head sections [Fig. 5(c)]. In the alcohol group, significant positive staining was observed in the trabecular bone, whereas positive staining was significantly reduced in the [email protected] group. Furthermore, a semiquantitative statistical analyses of the immunohistochemical staining results was performed [Fig. 5(d)]. The levels of cleaved caspase-3 in the Alcohol group were significantly higher compared with that in the control group; however, no significant differences were observed in comparison with the hydrogel group. Meanwhile, the cleaved caspase-3 levels in the [email protected] group were significantly lower compared with that in the alcohol group. These findings suggest that the nanocomposite hydrogel effectively inhibit the alcohol-induced MSC apoptosis.Osteopontin (OPN) and osteocalcin (OCN) are osteogenic markers expressed in bone marrow MSCs during osteogenic differentiation and calcium mineralization. We performed immunohistochemical staining for OPN [Fig. 5(e)] and OCN [Fig. 5(f)] to explore the osteogenic activity in the rat femoral head. Figures 5(g) and 5(h) show the results of the statistical analysis of the immunohistochemical staining results of OPN and OCN, respectively. The OPN and OCN levels in the alcohol group were significantly lower compared with that in the control group. These results indicate that exposure to alcohol significantly inhibits the formation of new bone in the femoral head, thereby further aggravating ONFH. However, the levels of OPN and OCN in the [email protected] group were significantly higher compared with that in the alcohol group. These results suggest that the nanocomposite hydrogel can significantly improve osteogenic activity in the femoral head of alcohol-induced ONFH rats, thus promoting the repair of ONFH-associated lesions. In conclusion, our findings demonstrate that the nanocomposite hydrogels can significantly inhibit alcohol-induced apoptosis of MSCs, promote osteogenic activity in the femoral head, and enhance the repair of necrotic sites during treatment of alcohol-induced ONFH.