Piezoelectric chitosan nanoparticles and their use in biomedical applications

Abstract

It forms an object of the present invention chitosan piezoelectric nanoparticles (ChNPs), methods to prepare them and their use.

Description

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[0003] PIEZOELECTRIC CHITOSAN NANOPARTICLES AND THEIR USE IN BIOMEDICAL APPLICATIONS BACKGROUND

[0004] Nanomaterials have received extensive attention and developed rapidly due to their special physical and chemical properties. In the biomedical field, nanomaterials show broad prospects in drug delivery, diagnostic imaging, tissue engineering and biosensing due to their special surface properties, size effects and structural characteristics. In terms of drug delivery, nanoparticles have a large specific surface area and surface activity and can be used as drug carriers to achieve directional delivery of drugs in the body; on the other hand, the special properties of nanoparticles themselves can also be used to achieve regulatory effects on cells and tissues.

[0005] Bioelectricity in the body is an important part of the regenerative microenvironment and a way for tissues to respond to external stimuli and regulate cell fate.

[0006] The piezoelectric effect refers to the generation of microcurrents by materials under exogenous mechanical vibrations, polarization and other stimuli. The unique ability of piezoelectric materials to generate electricity spontaneously has attracted widespread interest in the medical field. In addition to the ability to convert mechanical stress into electrical energy, piezoelectric materials offer the advantages of high sensitivity, stability, accuracy and low power consumption.

[0007] A variety of piezoelectric materials with piezoelectric effects have been described so far. Piezoelectric materials are classified into organic and inorganic. Among the inorganic, there are lithium tantalate (LiTaCh), lithium niobate (LiNbCh) and barium titanate (BaTiO,). However, these metal materials can cause toxicity to the human body.

[0008] Biodegradable options based on FDA-approved materials like chitosan have been explored in the form of piezoelectric films. As an example, CN110025818 describes chitosan piezoelectric films. While these films show promise for macroscopic applications, they are not suitable for subcellular-level interaction or localized therapeutic delivery due to their shape and scale.

[0009] Nanoparticles with piezoelectric properties can combine the nanosizedP1825PC00

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[0011] effect and the piezoelectric effect and have great application potential in the biomedical field.

[0012] CN115382013 discloses nanoparticles with piezoelectric properties having a core of BaTiO, and a coating of graphene oxide.

[0013] Hassani et al., in Investigation of the effect of chitosan nanoparticles synthesis method on their anticancer activity 2023, Int J Nanoscie. vol. 22, no. 01, describe superficially charged chitosan nanoparticles having a mean diameter of 420 nm being effective as anticancer agent, without mentioning any piezoelectric activity.

[0014] However, in films or fibers, the molecular or crystalline structure can be organized during their synthesis, allowing for the alignment of dipoles, which is essential for piezoelectricity. This means that a piezoelectric activity observed in films does not obviously translate into a piezoelectric activity in nanoparticles. The nanoscale structure can introduce quantum confinement effects, which alter the electronic properties of the material, potentially reducing its ability to generate electric charge under stress. Thus, while films and fibers can maintain the necessary structural integrity for piezoelectricity, nanoparticles often lose this ability due to disordered dipole orientation and surface effects (Spitzer, N.C. et al., BioEssays 2000, 22, 811).

[0015] It remains a strong need for biocompatible nanoparticles showing piezoelectric activity.

[0016] DESCRIPTION

[0017] In an embodiment, chitosan piezoelectric nanoparticles (ChNPs), methods to prepare them and their use are described.

[0018] DRAWINGS DESCRIPTION

[0019] Figure 1: (A) SEM image of chitosan nanoparticles, representative picture;

[0020] (B) Topographic map of the chitosan nanoparticles in AFM mode (without voltage application); (C) Topographic map of a single chitosan nanoparticle with the respective profilometric graph (D) obtained in AFM mode; (E) PFM of the same nanoparticle of (B), with the respective PFM profile (F) showing the displacement in response to electrical stimulation.

[0021] Figure 2: Cytocompatibility test (WST - 1) of ChNPS on patient-derivedP1825PC00

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[0023] glioblastoma cells (A, B) and on human astrocytes (C).

[0024] Figure 3: Calcium imaging during US stimulation, in presence or absence of ChNPs. (A) Time-lapse images at t = 0, 15, 30, 45, and 60 min. (B) Representative AF / F0 traces relative to Ca2+imaging time-lapses of hNSCs stimulated by US or US + ChNPs.

[0025] Figure 4: (A) Confocal fluorescence microscopy imaging of nuclei and ki-67 in Control, ChNPs, US, and US + ChNPs. (B) Increase in percentage of Ki-67-positive cells upon chronic piezoelectric stimulation.

[0026] Figure 5: (A) Confocal fluorescence microscopy imaging of nuclei and p53 in Control, ChNPs, US, US + ChNPs. (B) Decrease in percentage of p53-positive cells upon chronic piezoelectric stimulation.

[0027] Figure 6: (A) SEM image of PLLA nanoparticles, representative picture; (B) Topographic map of a single PLLA nanoparticle with the respective profilometric graph (C) obtained in AFM mode; (D) PFM of the same nanoparticle of (B), with the respective PFM profile (E) showing the displacement in response to electrical stimulation. No significant difference between the background and the NPs has been observed.

[0028] DETAILED DESCRIPTION

[0029] Objects of the present invention are piezoelectric chitosan nanoparticles (ChNPs) and method to prepare them.

[0030] In an embodiment, said ChNPs are for use in biomedical applications. In an embodiment, said ChNPS are for use in the treatment of tumors. In an embodiment, said chitosan nanoparticles have a diameter comprised in the range 320 - 400 nm as evaluated by Dynamic Light Scattering (DLS) using a Nano Z-Sizer 90 (Malvern), a poly dispersity index comprised in the range 0.230 - 0.330, a Zeta potential comprised in the range of 2 - 14 mV.

[0031] In an embodiment, said chitosan nanoparticles have a diameter of 362 ± 15 nm.

[0032] In an embodiment, the poly dispersity index of said ChNPs is 0.284 ± 0.035.P1825PC00

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[0034] In an embodiment, the Zeta potential of said ChNPs 8.25 ± 4.12 mV.

[0035] To evaluate the piezoelectric capability of the ChNPs according to the present invention, an INRiM metrological atomic force microscope equipped with a conductive PFM-specific tip (ASYELEC.01-R2 from Oxford Instruments) has been used. Experimental parameters for PFM measurements include an applied voltage of 1 V, scan size of 5 pm, scan rate of 0.6 Hz, scan speed of 7.15 pm / s, and tip oscillation frequency of 75 kHz. The piezoelectric coefficient d33 was derived by characterizing the individual particles in PFM mode and calculating the deformation value at the center of the single particles. An average d33 coefficient of 37.29 ± 4.45 pm / V was obtained.

[0036] The method to synthesize ChNPs comprises the following steps:

[0037] - preparation of an acid aqueous solution of chitosan at pH in the range of 2.3 and 2.6;

[0038] - preparation of an oil phase comprising surfactants, wherein said oil phase is prepared using dodecane and at least one surfactant selected from the group comprising Tween 80, Tween 85, Pluronic F68, Lecithin, Span 80, preferably Tween 85;

[0039] - adding drop by drop said aqueous solution of chitosan into said oil phase under continuous homogenization, obtaining an emulsion; - adding to said emulsion an aqueous basic solution at pH in the range of 13.4 and 13.9, wherein said basic solution comprises KOH; - removing the oil phase;

[0040] - centrifuging the aqueous phase, discard the supernatant, and wash the pellet resuspending it into water, said pellet comprising the chitosan nanoparticles.

[0041] In an embodiment, said acid solution is an acetic acid solution. In an embodiment, said acid solution is a 2.5% - 7.5% (v / v) acetic acid solution.

[0042] In an embodiment, said chitosan acid solution is a 0.5 - 2.0% chitosan solution.

[0043] In an embodiment, said oil phase is prepared with a ratio of dodecane to Tween 85 in the 7:1 - 13:1 range.

[0044] In an embodiment, said KOH solution is a 50% KOH solution.P1825PC00

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[0046] In an embodiment, said aqueous chitosan solution is vortexed for 5-10 minutes to ensure complete dissolution, after which it is left to rest for 10-30 min, allowing any bubbles to dissipate.

[0047] In an embodiment, said homogenization step is carried out at 15,000 RPM using T 10 basic ULTRA-TURRAX®.

[0048] The here proposed method allows to generate piezoelectric chitosan nanoparticles without the need for external factors such as poling or electro spinning.

[0049] Chitosan piezoelectric nanoparticles (ChNPs) represent a breakthrough in biomedical applications, offering several advantages over current materials, including piezoelectric chitosan films:

[0050] • Cell targeting and subcellular interaction: Unlike films, nanoparticles can penetrate tissue barriers and interact directly with cells or subcellular components, providing precise therapeutic effects.

[0051] • Enhanced biocompatibility: Chitosan is an FDA-approved, biodegradable, and biocompatible material, making the nanoparticles safer for long-term medical use, compared to non-biodegradable materials.

[0052] • Localized effects: The piezoelectric properties at the nanoscale allow for targeted electrical or mechanical stimulation, enhancing therapeutic outcomes such as neural tissue regeneration or cancer treatment.

[0053] • Broader applicability: The small size of the nanoparticles enables their integration into drug delivery systems or injectable formulations, which is challenging with films or other macroscopic materials.

[0054] Therefore, ChNPs combine the desirable properties of chitosan— biodegradability, biocompatibility, and FDA approval — with elevated piezoelectric effects at the nanoscale, enabling applications in advanced medical therapies and regenerative medicine. Compared to prior solutions, the nanoparticles provide a more versatile and safer approach for exploiting piezoelectricity in biological contexts.

[0055] Interestingly, the here provided data shown that chronic exposure to piezoelectric stimulation by using the ChNPs according to the present invention advantageously decrease expression of the proliferation marker Ki67 and increase the expression of the pro-apoptotic protein p53 on patient-derived glioblastoma cells. The effects are observed with exposure to ChNPs only,P1825PC00

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[0057] without the presence of any drugs.

[0058] Moreover, it is here shown an increased neuronal activity in human neural stem cells stimulated with ultrasound and ChNPs (US + ChNPs), wherein said activity is not elicited by US in the absence of ChNPs.

[0059] It forms a further object of the present invention ChNPs for use in the treatment of tumors.

[0060] It forms a further object of the present invention ChNPs for use in promoting osteogenesis, angiogenesis, and chondroblast differentiation to achieve bone repair.

[0061] EXPERIMENTAL SECTION

[0062] The following examples are merely intended to show some embodiments of the present invention and are not to be intended as limiting it in any way. The scope of the invention is defined by the appended claims.

[0063] Example 1: ChNPs synthesis

[0064] 0.5-2.0% chitosan solutions were obtained in 2.5-7.5% (v / v) acetic acid solutions. The mixtures are then vortexed for 5-10 minutes to ensure complete dissolution, after which they are left to rest for 10-30 min, allowing any bubbles to dissipate. 25 ml of the oil phase is then prepared with a ratio of dodecane to Tween 85 in the 7:1-13:1 range. Subsequently, 3.5-7.5 ml chitosan solution was added drop by drop to the oil phase while continuously homogenizing the mixture at 15,000 RPM using T 10 basic ULTRA-TURRAX®. The homogenization is carried out both during the addition of the chitosan solution and for an additional two min after this step.

[0065] Subsequently, the emulsion is treated with 5 ml of a 50% potassium hydroxide (KOH) solution (prepared with 50 g of KOH in 50 ml of water). After the addition of KOH, the mixture undergoes phase separation. The oil phase is removed using a glass pipette. The remaining aqueous phase is centrifuged at 11200 RCF for 15 min (Hettich®Universal 320 / 320R centrifuge). After centrifugation, the supernatant is discarded, and the pellet is resuspended in fresh Milli-Q water. This washing process is repeated six times, then pellet is resuspended in 4-5 ml of water and sonicated (FisherbrandTM Q125 Sonicator; 35% amplitude) for 40 min on ice to avoid heating.P1825PC00

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[0067] Example 2: ChNPs characterization

[0068] The nanoparticles obtained as detailed in Example 1 were analyzed at Dynamic Light Scattering (DLS) to assess the average diameter, the polydispersity index, and the Zeta potential. The nanoparticle diameter is 362 ± 15 nm, the polydispersity index 0.284 ± 0.035, and the Zeta potential 8.25 ± 4.12 mV.

[0069] Using a Scanning Electron Microscopy (SEM) (Helios NanoLab 600i FIB / SEM, FEI), nanoparticles were observed, as shown in Fig.1A.

[0070] Afterward, the nanoparticles were analyzed for their piezoelectricity by using piezoresponse force microscopy (PFM; Fig. 1B-F). A droplet of diluted sample (50 pg / mL in Milli-Q water) was cast on a silicon wafer and allowed to dry. An INRiM metrological atomic force microscope equipped with a conductive PFM-specific tip (ASYELEC.01-R2 from Oxford Instruments) was used for both PFM and atomic force microscopy (AFM) measurements in tapping mode. Experimental parameters for PFM measurements include an applied voltage of 1 V, scan size of 5 pm, scan rate of 0.6 Hz, scan speed of 7.15 pm / s, and tip oscillation frequency of 75 kHz. The piezoelectric coefficient d33 was derived by characterizing the individual particles in PFM mode and calculating the deformation value at the center of the single particles; the measurement was repeated for 15 nanoparticles and then average d33 was calculated. An average d33 coefficient of 37.29 ± 4.45 pm / V was obtained.

[0071] Example 3: Biocompatibility test

[0072] The viability of glioblastoma patient-derived cells and immortalized human astrocytes incubated with ChNPs was evaluated using the WST-1 assay (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt in a premix electrocoupling solution, BioVision). Five concentrations were evaluated (0, 100, 200, 300, and 600 pg / mL; six replicates per concentration). 24 h after cell seeding (seeding density was 15000 cells / cm2), the medium was removed from each well and replaced with the medium containing the corresponding concentration of ChNPs. At 72 h of incubation, WST-1 assay was then performed. The culture medium was removed, and each well was treated with WST-1 solution (1:11 dilution in phenol red-free and HEPES-supplemented DMEM with 10% FBS). The plate was incubated for 40 min, after which 80 pL from each well was transferred to a 96-well plate for absorbanceP1825PC00

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[0074] reading. Absorbance was measured at 450 nm using a Victor3 microplate reader (PerkinElmer), and metabolic activity for each condition was normalized against the absorbance of the control cultures.

[0075] Results related to glioblastoma patient-derived cells after ChNPs treatments are summarized in Fig.2. Fig.2A shows the microscope images of the cells in different experimental conditions. No cell detachment was observed, not even when treating cells with the higher NP concentration (600 pg / mL). Accordingly, no significant change in cell viability (%) was detected with the WST-1 cytocompatibility text (Fig. 2B; ANOVA test; p> 0.05), indicating that ChNPs are highly biocompatible and support cell growth.

[0076] Viability of astrocytes after ChNPs treatments is shown in Fig. 2C.

[0077] Similarly, no significant change in cell viability (%) was detected with the WST-1 cytocompatibility text (ANOVA test; p > 0.05), confirming that ChNPs are highly biocompatible and support cell growth.

[0078] Example 4: Activation of intracellular calcium waves in neural stem cells Calcium imaging was performed to monitor the real-time neuronal activity of human neural stem cells (hNSCs) undergoing piezoelectric stimulation, triggered by ultrasound (US) as mechanical activator of piezoelectric particles, according to Marino, A. et al., ACS Nano 2015, 9, 7678.

[0079] The effects of mechanical stimulation alone using US with those mediated by US + ChNPs were compared. This analysis enabled assessing whether an increased calcium-mediated activation occurs during piezoelectric stimulation. Furthermore, calcium transients play a critical role in the regulation of many cell processes, including those related to neuronal differentiation and function (Spitzer, N.C. et al. BioEssays 2000, 22, 811). For this experiment, hNSCs from hindbrain (Y40060; Takara Bio Inc.), were seeded on laminin-coated 24-well plates (Ibidi) at 50% of confluence. hNSCs were cultured in RHB-A medium (Takara Bio Inc.) supplemented with 100 pg / m streptomycin (Gibco), 20 ng / m recombinant human epidermal growth factor (EGF, Peprotech), and 20 ng / m recombinant human fibroblast growth factor (FGF-2 / bFGF, Peprotech) at 37° in 5% CO2 fully humidified atmosphere. For US +ChNPs experimental class, hNSCs were pre-incubated for 24 h with 300 pg / m ChNPs. Cells were incubated with Fluo-4 AM (1 pM, Invitrogen) at 37°C for 40 min prior to US stimulation. US stimulations were performed using a KT AC-4000 device (Sonidel) equipped withP1825PC00

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[0081] a planar ultrasound transducer (20 mm diameter) operating at a power of 0.9 W / cm2, a frequency of 1 MHz, a burst rate of 0.5 Hz and a duty cycle of 10%. These specific US parameters were chosen to avoid significant temperature increases in the cell medium, even during prolonged stimulation. Cells were stimulated for 1 h; calcium imaging was started 10 min before the stimulation. The samples were then rinsed with PBS and incubated with phenol red-free DMEM supplemented with HEPES (25 mM, Thermo Fisher) for time-lapse fluorescence imaging using an Eclipse Ti-E confocal microscope (Nikon). Images were acquired at a rate of one every 10 s. Fluorescence intensities at different time points (F) were calculated as the mean value of pixels measured in the intracellular region of interest (ROI). The graphs in Fig. 3B show F normalized for the value of F at time t = 0s (F / F0) in the different experimental conditions (US and US + ChNPs). Representative images at t = 0, 15, 30, 45 and 60 min were selected for both experimental classes. Fig. 3A reports representative images of the time-lapse. In US, no significant cell activation is visible; conversely, when cells undergo the US + ChNPs stimulation, a significant response in terms of calcium waves is clearly detected.

[0082] Fig.3B reports the F / F0 traces. In the absence of ChNPs, the calcium signal remains constant throughout the stimulation period. Conversely, in the presence of ChNPs, different calcium waves in most of the cells can be observed after starting the stimulation (t = 10 min), indicating an increased cell activity. The imaging of the US class confirms that mechanical stimulation alone, in these experimental conditions, is not causing the activation of calcium signal.

[0083] Example 5: Anticancer effects of ChNPs-assisted piezoelectric stimulation in patient-derived glioblastoma cells

[0084] Immunofluorescence staining was carried out to evaluate the expression of the p53 pro-apoptotic protein and the Ki-67 proliferation marker in patient-derived GBM cells following 3 days of chronic piezoelectric stimulation (Ih of US per day using the same stimulation parameters specified for calcium imaging experiments).

[0085] Cell cultures were fixed using a 4% paraformaldehyde (PF A) solution in phosphate-buff ered saline (PBS) (4 °C, 25 min). Membrane permeabilization was achieved with 0.1% Triton X-100 (Sigma- Aldrich) in PBS at room temperature for 1 h, followed by blocking with 10% goat serum in PBS for 1 h at 37 °C. For immunostaining, the cultures were incubated with either a primary mouseP1825PC00

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[0087] monoclonal anti-p53 antibody (diluted 1:150, Abeam) or a primary rabbit IgG anti-Ki-67 antibody (diluted 1:150, Millipore) in 10% goat serum at 37 °C for 45 minutes. After three washes, the samples were treated with a secondary antibody solution containing TRITC-conjugated anti-rabbit IgG (1:250, Millipore) or FITC-conjugated anti-mouse IgG (1:250, Millipore) and counterstained with Hoechst 33342 (2 pg / ml, Invitrogen) for nuclear visualization (1 h treatment with the staining solution). Post-staining, the cultures were washed three times with PBS before being imaged using a confocal laser scanning microscopy (CLSM) system (C2s, Nikon). Fig. 4A shows representative images obtained under different experimental conditions. Ki-67 fluorescence is highly expressed in the Control group as well as in samples treated with ChNPs or US alone. In contrast, lower Ki-67 expression is observed in the experimental group subjected to chronic piezoelectric stimulation (US + ChNPs). These qualitative observations align with the quantitative data presented in the histogram in Fig. 4B, which reports the percentage of Ki-67-positive cells for each experimental group.

[0088] Quantitatively, Ki-67 expression levels are relatively high in the control group (48.83 ± 4.22%) and in groups treated with ChNPs alone (48.57 ± 5.56%) or US alone (52.34 ± 4.25%), with approximately half of the analyzed cells in the proliferative phase. In contrast, the group treated with the chronic piezoelectric stimulation (US + ChNPs) shows a significantly lower Ki-67 expression (17.33 ± 5.06%), representing a 2.82-fold reduction compared to the Control group. Fig.

[0089] 5A displays representative images highlighting p53 expression under different experimental conditions (Control, ChNPs, US, and US + ChNPs). Immunofluorescence analysis reveals that p53 levels, a marker of tumor suppression and cellular stress response, are notably increased in cells treated with the combination of US and ChNPs compared to all other groups. In the control group and those treated with ChNPs alone or US alone, p53 fluorescence is minimal, suggesting a lower activation of tumor-suppressive pathways under these conditions. The increased p53 expression in the US + ChNPs group is evident both qualitatively in Fig.5A and quantitatively in Fig.5B, which presents the percentage of p53-positive cells for each experimental class. Quantitatively, p53 expression levels are low in the control group (3.63 ± 1.42%) as well as in the groups treated with ChNPs alone (2.70 ± 1.44%) or US alone (2.32 ± 1.55%).

[0090] In contrast, cells exposed to the combined treatment of ChNPs and US exhibit significantly elevated p53 levels (17.65 ± 4.45%), demonstrating a remarkable increase in the activation of tumor suppressor mechanismsP1825PC00

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[0092] (approximately 4.86-fold higher than the control group).

[0093] These results strongly suggest that chronic piezoelectric stimulation via US + ChNPs not only inhibits cell proliferation, as evidenced by Ki-67 downregulation, but also effectively promotes tumor suppression pathways through enhanced p53 expression.

[0094] This study represents the first demonstration of reduced ki-67 expression and enhanced p53 activation induced by piezoelectric stimulation using FDA-approved nanomaterials, specifically ChNPs. This result highlights the potential of combining US and biocompatible, regulatory-approved, biodegradable, bioabsorbable materials for inducing antiproliferative and apoptotic pathways in the context of minimally invasive anticancer therapies.

[0095] Example 6: Non-deriv ability of emerging piezoelectric properties in nanoparticle form: PLA as supporting proof (comparative)

[0096] While poly(lactic acid) (PLA) materials are widely known for their piezoelectric properties in films (Ben Achour, M. A. et al., ACS Appl Polym Mater 2023, 5, 9761) and electrospun scaffolds (Schonlein, R. et al., Mater Des 2024, 237, DOI 10.1016 / j.matdes.2023.112525; Hong, S.K. et al., Polymers (Basel) 2024, 16, DOI 10.3390 / polyml6030347) these properties do not directly translate to nanoparticles. This distinction is critical because, at the nanoparticle level, the emergence of piezoelectric behavior is not inherently expected. PLA, therefore, serves as a useful comparative example in supporting the inventive nature of the here proposed method. We fabricated poly-l-lactic acid (PLLA) nanoparticles and characterized them with PFM to study their piezoelectric properties.

[0097] To fabricate PLLA nanoparticles, we dissolved 20 mg PLLA and 2 mg propylene glycol in 2 mL of a solution of 96% ethanol (0.62 mL) and chloroform (1.38 mL) and then dripped in 3.8 mL of a 70% ethanol solution under stirring (300 rpm at 22° C). Subsequently, we heated the solution under stirring (300 rpm at 60° C) until the solvent evaporated. An average diameter of 306 ± 11 nm and a polydispersity index of 0.217 ± 0.02 were measured by DLS. SEM imaging of the PLLA nanoparticles is reported in Fig. 6A. PFM was performed on PLLA NPs using the same experimental procedures described for chitosan NPs. Data are reported in Fig. 6B-E. The NPs showed no significant piezoelectric properties (0.11 ± 0.07 pm / V)

Claims

P1825PC00- 12- CLAIMS1. Chitosan nanoparticles (ChNPs) having a diameter comprised in the range 320 - 400 nm as evaluated by Dynamic Light Scattering (DLS) using a Nano Z-Sizer 90 (Malvern), a polydispersity index comprised in the range 0.230 - 0.330, a Zeta potential comprised in the range of 2 - 14 mV.

2. ChNPs according to claim 1, having a diameter of 362 ± 15 nm, a polydispersity index of 0.284 ± 0.035, a Zeta potential of 8.25 ± 4.12 mV.

3. ChNPs according to claim 1 or 2, having a d33 coefficient of 37.29 ± 4.45 pm / V, as evaluated by a metrological atomic force microscope.

4. A method to obtain ChNPs comprising:- preparation of an acid aqueous solution of chitosan at pH in the range of 2.3 and 2.6;- preparation of an oil phase comprising surfactants, wherein said oil phase is prepared using dodecane and at least one surfactant selected from the group comprising Tween 80, Tween 85, Pluronic F68, Lecithin, Span 80, preferably Tween 85;- adding drop by drop said aqueous solution of chitosan into said oil phase under continuous homogenization, obtaining an emulsion; - adding to said emulsion an aqueous basic solution at pH in the range of 13.4 and 13.9, wherein said basic solution comprises KOH;- removing the oil phase;- centrifuging the aqueous phase, discard the supernatant, and wash the pellet resuspending it into water, said pellet comprising the chitosan nanoparticles.

5. The method according to claim 4, wherein said acid solution is an acetic acid solution.

6. The method according to claim 4 or 5, wherein said chitosan acid solution is a 0.5 - 2.0% chitosan solution.

7. The method according to any one of the claims 4-6, wherein said oil phase is prepared with a ratio of dodecane to surfactant in the 7:1 - 13:1 range.

8. ChNPs according to any one of the claims 1-3, for use in biomedical applications.P1825PC00-13-9. ChNPs for use according to claim 8, wherein said application is tumor treatment.

10. ChNPs for use according to claim 8, wherein said application is in promoting osteogenesis, angiogenesis, and chondroblast differentiation to achieve bone repair.

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