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• Neurodegenerative diseases are a heterogeneous family of complex diseases characterized by neuronal loss and progressive degeneration of different areas of the nervous system. Alzheimer's disease (AD) is characterized by the presence of extracellular amyloid-β plaques and intracellular neurofibrillary tangles, accompanied by obvious neuron loss or damage [1–3]. Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra [4]. Despite tremendous advances in modern medicine [5–7], effective prevention or therapeutic strategies for neurodegenerative diseases such as AD and PD remain limited. Neurons are the most basic structural and functional units in the brain and their damage is the direct cause of neurodegeneration [8,9]. Microglia, as the resident glia cells of the brain, is well known to play significant roles by involving in inflammation of neurodegenerative diseases [10,11]. They display great potential as targets for therapeutic intervention.

  Accumulating studies have found that microRNAs (miRNAs) play a crucial role in regulating proliferation, differentiation, and death of cells in neurodegenerative disorders [12,13]. miRNAs are a class of highly conserved non-coding RNAs (~18–25 nucleotides in length) which regulate diverse biological processes by modulating gene expression at the posttranscriptional level [14]. A series of miRNAs have been proved to be involved in the pathogenesis of AD, one of the most popular neurodegenerative disorders [15]. miR-34a, which has been found to be important in the regulation of the neuronal cell cycle and apoptosis [16], has been reported to be deregulated in both the brain and peripheral blood of AD patients [17,18]. Thus, miRNAs are increasingly associated with neurodegenerative diseases and are emerging as promising cell-based therapeutic drugs. However, naked miRNAs can hardly enter the cells directly due to physiological barriers, which make nanocarriers for miRNA delivery to cells, especially brain cells a promising treatment strategy for neurodegenerative disorders.

  Polymer nanoparticles (NPs) have been widely used as drug delivery systems because they are able to not only provide controlled or sustained drug release at appropriate or therapeutic concentrations, but also improve the pharmaceutical parameters of the drug such as bioavailability and half-life [19–23]. Among them, chitosan (CS) NPs stand out for their nontoxicity, neurotoxicity, biodegradability, and physiologically stability [24–26]. The properties of nontoxicity and neurotoxicity are very important for the drug delivery of brain cells as brain cells are extra sensitive to toxicity and may lead to irreversible brain damage [27,28], which is the biggest challenge for the brain cell-targeted drug delivery. In this study, chitosan nanoparticles were prepared to work as the delivery carriers of miR-34a to the neuronal and microglia cells. The delivery efficiency was assessed from various aspects such as the miRNA encapsulation efficiency, the controlled releasing behavior, the toxicity to the cells, and the transfection efficiency.

  CS NPs were prepared by ionic gelation method with minor modifications of the method reported by Mohammadi-Samani [29]. In brief, CS solution at a concentration of 2.5 mg/mL was prepared by dissolving CS into 1% glacial acetic acid solution. A certain amount of tripolyphosphate (TPP) was weighed to prepare a 1 mg/mL TPP solution. The TPP solution was slowly added to CS solution dropwise under magnetic stirring with CS/TPP mass ratio of 4:1, followed by another 10 min of stirring at 500 rpm. Then the CS NPs were collected by dialysis, centrifuged at 13,000 rpm for 10 min, and stored at −20 ℃. Zetasizer Nano ZS system (Malvern Instruments Worcestershire, ZEN3700, UK) was utilized to measure the particle size, size distribution, and zeta potential of the NPs. The particle morphology was observed by scanning electron microscopy (SEM). CS NPs were subjected to Fourier transform infrared (FTIR) analyses with a FTIR spectrometer (Bruker Tensor 27, Bruker Optik GmbH, Ettlingen, Germany) at frequency range of 400–4000 cm⁻¹. Different amount of miR-34a mimic was added to prepare miR-34a-loaded CS NPs. To confirm miR-34a was encapsulated in CS NPs and assess the stability of the complexation, agarose gel retardation assay was carried out. Entrapment efficiency (EE) of miR-34a was calculated by indirect method with the following equation: EE = (weight of loaded drug/weight of total drug) × 100%.

  The in vitro release study of miR-34a from miRNA-loaded CS NPs was carried out in phosphate buffer saline (PBS) (pH 7.4) and hydrate chloride buffer (pH 1.2), respectively. The percentage of cumulative miR-34a released was then presented by the ratio of the cumulative amount of miR-34a released at each time interval to the initial amount of the miR-34a encapsulated in the NPs. With the optimal conditions, different concentrations of miR-34a-loaded CS NPs were prepared to assess their toxicity to mouse hippocampus neuronal HT22 cells and mouse microglia BV2 cells. Their transfection efficiency in the two cell lines were examined at the same time.

  After the optimization of various conditions such as pH, weight ratio of CS and TPP, and dropping speed of TPP, CS NPs were synthesized successfully by ionic gelation method. They displayed nearly spherical shape with smooth surfaces (Fig. 1A). Their size distribution and positive zeta potential (13.3 ± 2.9 mV) of CS NPs were shown in Figs. 1B and C. Particle size and surface charges are critical determinants for drug delivery carriers as they can influence the drug entrapment efficiency and transfection efficiency [30,31]. In details, the size of CS NPs determines the way of NPs entering the cells and the positive charges is the key property to encapsulate drugs with negative charges such as nucleic acid drugs [32–34].

  Figure 1

  

  Figure 1.  (A) SEM image, (B) size distribution curve, and (C) zeta potential of CS NPs. (D) FTIR spectra of CS, CS NPs and TPP.

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  To confirm the complexation reaction during the CS NP formation, CS, CS NPs and TPP were subjected to FTIR assay. As shown in Fig. 1D, CS spectrum demonstrated its characterization peaks at 3356 cm⁻¹ (-OH and -NH₂ stretching), 1624 cm⁻¹ (amide Ⅰ band), 1516 cm⁻¹ (N-H deformation) and 1076 cm⁻¹ (C-O stretching). After particle formation, the amide Ⅰ peak at 1624 cm⁻¹ reduced obviously and the amide Ⅱ peak at 1516 cm⁻¹ in CS shifted to 1544 cm⁻¹ in CS NPs, verifying the cross-linking of amino groups with phosphate. Moreover, the peak in the CS at 3356 cm⁻¹ widened and shifted to 3371 cm⁻¹ in the CS NPs, indicating enhanced hydrogen bonding. These changes were consistent with previous studies [34–36] and enough to prove the successful ionic cross-linking reaction. miR-34a was added to prepare miR-34a-loaded CS NPs. To confirm the encapsulation of miR-34a in CS NPs and examine the stability of miR-34a/CS NPs complexation, the agarose-gel retardation assay was carried out. As shown in Fig. 2A, obvious miRNA migration appeared after sodium dodecyl sulfate (SDS) treatment of the complexation and miRNA migration was completely retarded after encapsulation with the miR-34a/CS mass ratio of 100:1, 200:1, 400:1, which verified the encapsulation of miR-34a in CS NPs and indicated good stability of the complexation. However, miRNA migration was partially retarded with the miR-34a/CS mass ratio of 50:1, suggesting the miRNA dose was beyond the loading capacity of the CS NPs. Moreover, the band of naked miR-34a was not as dark as the miR-34a-loaded CS NPs containing equal amount of miR-34a (Fig. 2A), which suggest the complexation protected miR-34a from degradation. In addition, miR-34a-loaded CS NPs showed similar shape and size with blank CS NPs (Fig. 2B). And they displayed nearly neutral in surface charge (Fig. 2C), which means the loading of miR-34a neutralized the positive charges of blank CS NPs. Blank CS NPs are positively charged and miRNAs are negatively charged nucleic acid drugs. Cationic CS NPs are supposed capable to bind and condense RNAs by electrostatic interactions, which can avoid degradation of RNAs by RNase and shield the electrostatic repulsion between RNAs and the negatively charged cell membrane [37,38].

  Figure 2

  

  Figure 2.  (A) Agarose gel retardation assay of miR-34a-loaded CS NPs. Bands: (1) naked miR-34a; (2) blank CS NPs; (3) synthesized miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 50:1; (4) SDS-treated miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 50:1; (5) synthesized miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 100:1; (6) SDS-treated miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 100:1; (7) synthesized miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 200:1; (8) SDS-treated miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 200:1; (9) synthesized miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 400:1; (10) SDS-treated miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 400:1. (B) SEM image. (C) Zeta potential of miR-34a-loaded CS NPs.

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  Entrapment efficiency is one of the most important properties of drug delivery carriers. The entrapment efficiency of CS NPs with different doses of miR-34a addition was assessed. As shown in Fig. 3A, the entrapment efficiency at the CS/miR-34a ratio of 100:1 was significantly higher than other ratios. The ratio of 50:1 displayed extra low efficiency probably because the overdosed miR-34a was not able to be entrapped into CS NPs, which was in agreement with the above results of agarose-gel retardation assay. Drug release profile is another key parameter of drug delivery vectors. Drug release from particles is primarily realized by three ways: (a) Desorption from the surface of particles (burst release), (b) diffusion through the particle structure, and (c) release due to polymer erosion [39,40]. Release profiles of miR-34a in two solutions with different pH (7.4 and 1.2) were explored within 96 h. In PBS (pH 7.4), miR-34a-loaded CS NPs showed an approximate 15.3% initial burst within 1.5 h, followed by steady miR-34a release of 52.0% within 12 h and slower release afterwards. The accumulated miR-34a release then gradually reached 73.4% within 96 h (Fig. 3B). However, the accumulated miR-34a release in hydrochloric acid buffer (pH 1.2) within 96 h was < 10% (Fig. 3B), which might result from miR-34a degradation in the acidic environment.

  Figure 3

  

  Figure 3.  (A) Entrapment efficiency of miR-34a-loaded CS NPs with different mass ratio of CS to miR-34a. (B) miR-34a release profile from miR-34a-loaded CS NPs in media at pH of 1.2 and 7.4. Data are presented as mean ± standard deviation (SD) (n = 3). ****P < 0.0001.

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  The cytotoxicity of miR-34a-loaded CS NPs to mouse neuronal HT22 cells was evaluated by MTT assay. As shown in Fig. 4A, the percentages of viable cells were greater than 90% after being treated with miR-34a-loaded CS NPs with miR-34a concentrations of 50, 100, and 200 nmol/L, respectively. miR-34a-loaded Lipo2000 demonstrated the similar viability. The high cell viability at these concentrations indicated that CS NPs exerted little cytotoxicity to neuronal cells. Meanwhile, the transfection efficiency was recorded. miR-34a-loaded CS NPs showed the highest transfection efficiency at miR-34a concentration of 100 nmol/L and this was significantly higher than control Lipo2000 (Fig. 4B). With the optimal miR-34a concentration, the fluorescence signal was detected in HT22 cells exposed to miR-34a-loaded CS NPs. The percentage of cells with fluorescence was significantly higher when exposed to miR-34a-loaded CS NPs than control miR-34a-loaded Lipo2000 at all time points (Fig. 4C). The results also showed that the transfection efficiency rose quickly in 3 h but kept stable in the following 3 h. Taken together, miR-34a-loaded CS NPs were endocytosed into neuronal cells without obvious cytotoxicity.

  Figure 4

  

  Figure 4.  (A) Viability and (B) transfection efficiency of HT22 cells after treated with different concentrations of miR-34a-loaded CS NPs or miR-34a-loaded Lipo2000. (C) Transfection efficiency of HT22 cells transfected with miR-34a-loaded CS NPs or miR-34a-loaded Lipo2000 within 6 h. Data are presented as mean ± SD (n = 4). ***P < 0.001, ****P < 0.0001; ^##P < 0.01. ns, P > 0.05.

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  The cytotoxicity of miR-34a-loaded CS NPs to mouse microglia BV2 cells was studied by MTT assay. As shown in Fig. 5A, the percentages of viable cells were over 90% after being exposed to miR-34a-loaded CS NPs at miR-34a concentrations of 50, 100, and 200 nmol/L. miR-34a-loaded Lipo2000 demonstrated the similar viability. The high cell viability at these concentrations indicated that CS NPs exerted little cytotoxicity to microglia cells. The transfection efficiency was recorded at the same time. miR-34a-loaded CS NPs showed the highest transfection efficiency (over 60%) at miR-34a concentration of 100 nmol/L and this was significantly higher than control Lipo2000 (Fig. 5B). With the optimal miR-34a concentration, the fluorescence signal was detected in BV2 cells exposed to miR-34a-loaded CS NPs. The percentage of cells with fluorescence was significantly higher when exposed to CS NPs than control Lipo2000 at the time points of 1 h and 6 h (Fig. 5C). The transfection efficiency increased quickly in the beginning 3 h and became steady in the following 3 h, which happened in cells exposed to both miR-34a-loaded CS NPs and miR-34a-loaded Lipo2000 (Fig. 5C). Collectively, miRNAs were successfully delivered into microglia cells without significant cytotoxicity.

  Figure 5

  

  Figure 5.  (A) Viability and (B) transfection efficiency of BV2 cells after treated with different concentrations of miR-34a-loaded CS NPs or miR-34a-loaded Lipo2000. (C) Transfection efficiency of BV2 cells transfected with miR-34a-loaded CS NPs or miR-34a-loaded Lipo2000 within 6 h. Data are presented as mean ± SD (n = 4). P < 0.05, **P < 0.01, ****P < 0.0001; ^##P < 0.01.

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  In conclusion, this study demonstrated that spherical and mono dispersed CS NPs can be prepared by an ionic gelation method. With excellent safety and high miRNA delivery efficiency, CS NPs are considered prospective carriers for the miRNA delivery to both neuronal and glia cells, which may further contribute to the early interference and treatment of neurodegenerative diseases. However, it is a long way to bring chitosan nanoparticles to clinical applications in neurodegenerative diseases. In another word, there are many difficulties to overcome such as higher transfection efficiency of the vectors, the capability of CS NPs to cross the blood-brain barrier, the drawbacks of the administration way, and the side effects of untargeted delivery. Recently, nose to brain delivery has appeared as a novel administration route for brain drug delivery and it presents a series of unique advantages such as the direct delivery to minimize systemic exposure and the obviously improved utilization rate of therapeutic agents. Above all, a number of efforts are required to make in these aspects.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Lian Jin: Writing – original draft, Funding acquisition, Conceptualization. Juan Zhang: Writing – original draft, Methodology, Investigation. Libo Nie: Writing – review & editing, Supervision. Yan Deng: Writing – review & editing, Supervision. Ghulam Jilany Khana: Project administration, Investigation. Nongyue He: Writing – review & editing, Project administration, Funding acquisition.

  Acknowledgments

  The work was supported financially by the NSFC (Nos. 62075098 and 62071119) the National Key Research and Development Program of China (Nos. 2017YFA0205301 and 2018YFC1602905).

  
  
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  Figure 1  (A) SEM image, (B) size distribution curve, and (C) zeta potential of CS NPs. (D) FTIR spectra of CS, CS NPs and TPP.

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  Figure 2  (A) Agarose gel retardation assay of miR-34a-loaded CS NPs. Bands: (1) naked miR-34a; (2) blank CS NPs; (3) synthesized miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 50:1; (4) SDS-treated miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 50:1; (5) synthesized miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 100:1; (6) SDS-treated miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 100:1; (7) synthesized miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 200:1; (8) SDS-treated miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 200:1; (9) synthesized miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 400:1; (10) SDS-treated miR-34a-loaded CS NPs with the mass ratio of CS to miR-34a of 400:1. (B) SEM image. (C) Zeta potential of miR-34a-loaded CS NPs.

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  Figure 3  (A) Entrapment efficiency of miR-34a-loaded CS NPs with different mass ratio of CS to miR-34a. (B) miR-34a release profile from miR-34a-loaded CS NPs in media at pH of 1.2 and 7.4. Data are presented as mean ± standard deviation (SD) (n = 3). ****P < 0.0001.

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  Figure 4  (A) Viability and (B) transfection efficiency of HT22 cells after treated with different concentrations of miR-34a-loaded CS NPs or miR-34a-loaded Lipo2000. (C) Transfection efficiency of HT22 cells transfected with miR-34a-loaded CS NPs or miR-34a-loaded Lipo2000 within 6 h. Data are presented as mean ± SD (n = 4). ***P < 0.001, ****P < 0.0001; ^##P < 0.01. ns, P > 0.05.

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  Figure 5  (A) Viability and (B) transfection efficiency of BV2 cells after treated with different concentrations of miR-34a-loaded CS NPs or miR-34a-loaded Lipo2000. (C) Transfection efficiency of BV2 cells transfected with miR-34a-loaded CS NPs or miR-34a-loaded Lipo2000 within 6 h. Data are presented as mean ± SD (n = 4). P < 0.05, **P < 0.01, ****P < 0.0001; ^##P < 0.01.

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