Development and use of RBD nanoparticle-loaded dissolving microneedles

Abstract

The invention relates to a dissolving microneedle containing nanoparticles loaded with a Receptor-Binding Domain (RBD).

Description

[0001] DESCRIPTION

[0002] DEVELOPMENT AND USE OF RBD NANOPARTICLE-LOADED DISSOLVING MICRONEEDLES

[0003] Technical Field

[0004] The invention relates to a dissolving microneedle containing nanoparticles loaded with a Receptor-Binding Domain (RBD).

[0005] Prior Art

[0006] The entry of SARS-CoV-2 into the cell is mediated by binding to angiotensinconverting enzyme 2 (ACE2), a SARS-CoV-2 receptor found on the outer membranes of cells in the lungs, arteries, heart, kidney and intestines. Entry of the coronavirus into the host cell is a complex, multi-step, highly regulated process involving multiple processing steps of the S protein. The entire spike protein S is cleaved into SI and S2 subunits, and SI also participates in receptor recognition by recruiting the receptor binding domain (RBD). The S protein has been investigated by various groups using different techniques and it has been reported that the RBD region of the S protein is key for receptor binding. [1]

[0007] In vitro binding measurements showed that SARS-CoV-2 RBD binds to ACE2 with an affinity in the low nanomolar range, suggesting that RBD is a key functional component within the SI subunit responsible for SARS-CoV-2 binding by ACE2. [2]

[0008] RBD is therefore identified as the most likely target for the development of therapeutic virus binding inhibitors, induction of neutralizing antibodies and vaccine design. [3] Subunit vaccines are among the safest and most widely used vaccines and are highly effective against various infectious diseases such as hepatitis B, diphtheria, pertussis, tetanus and shingles in various age groups. Therefore, the development of an effective and safe subunit vaccine against SARS-CoV-2 will be an important step in controlling the COVID 19 pandemic. [4]

[0009] Subunit vaccines are generally safe and reduce the risks associated with pathogen challenge, virulence recovery and induction of harmful immune responses. However, these vaccines generally do not induce strong or long-lasting immune responses and require appropriate adjuvants and repeated doses. [3]

[0010] Nanoparticles are designed to allow efficient delivery where the antigen can be encapsulated on the surface or inside the carrier system. Thus, an encapsulated antigen is not easily degraded and release remains controlled at the target site for longer periods of time. Controlled antigen release prevents an exacerbated response and eliminates the need for repeated doses. [5]

[0011] Polymeric nanoparticles are colloidal systems with a wide size range (10-1000 nm). Polymeric nanoparticles have high immunogenicity and stability for efficient encapsulation and display of antigen that can be loaded into the core and conjugated to the surface. They also have controllable sizes and can be self-adjuvant and improve the efficiency of antigen uptake by antigen-presenting cells. [6]

[0012] Currently, many vaccines are administered intramuscularly and intradermally. However, there are several disadvantages, including pain and fear of needle sticks, the need for two doses, the need for trained healthcare professionals for vaccine administration, and the need for a cold supply chain. Recently, transdermal techniques for vaccines, including COVID-19, have been explored. Microneedle technology uses multiple microscopic projections from a plate that administers the vaccine in the form of a patch placed on the skin, allowing painless antigen administration with enhanced immune response. [7]

[0013] In the skin, antigens released from microneedles are taken up by tissue-resident antigen-presenting cells (APCs) that travel to the lymph nodes to initiate immune responses. The high abundance of APCs in the skin and effective targeting of these cells is critical for achieving protective immunity. These advantages make cutaneous vaccination more efficient than other routes of administration. Another advantage of microneedle vaccines is that the microneedles do not reach pain receptors in the skin, providing a virtually painless route of vaccine delivery. [8]

[0014] Dissolving microneedles are designed to encapsulate drugs or particles into a water- soluble polymer matrix and once applied to the skin, to dissolve completely with a dissolution time that varies according to the formulation. The dissolving microneedles do not leave any biohazardous sharps waste after use. Once inserted into the skin, drug formulations are released from the dissolving microneedle matrix over time, either slowly or rapidly depending on the polymer composition. [9]

[0015] The publication entitled Adjuvanted-SARS-CoV-2 Spike Protein-Based Microparticulate Vaccine Delivered by Dissolving Microneedles Induces Humoral, Mucosal, and Cellular Immune Responses in Mice, which is in the state of the art, describes the evaluation of the synergistic effects of a subunit microparticulate vaccine delivered using microneedles.

[0016] In the International Patent document numbered WO2022058764A1, which is in the known state of the art, skin patches for the diagnosis of immunological protection against SARS-COV-2 virus infection are mentioned.

[0017] In the publication document titled Development of Spike Receptor-Binding Domain Nanoparticles as a Vaccine Candidate Against SARS-CoV-2 Infection in Ferrets, which is in the known state of the art, it is mentioned that spike RBD- nanoparticles are an effective protein vaccine candidate against SARS-CoV-2.

[0018] When the existing studies in the art were examined, there was a need to develop a soluble microneedle containing RBD-loaded nanoparticles.

[0019] Objectives of the Invention

[0020] It is an object of the present invention to provide a dissolving microneedle comprising nanoparticles loaded with a receptor-binding domain (RBD).

[0021] Another object of the present invention is to provide a dissolving microneedle comprising nanoparticles loaded with an RBD, whereby the stability of the vaccine is increased, cold chain application and fear of injection is avoided.

[0022] Detailed Description of the Invention

[0023] The dissolving microneedle realized to achieve the objects of the present invention is shown in the accompanying figures.

[0024] These figures are;

[0025] Figure la: Graphical representation of the hydrodynamic diameter profiles of RBD nanoparticles used in the dissolving microneedle of the invention.

[0026] Figure lb: A graphical view of the zeta potential profiles of RBD nanoparticles used in the dissolving microneedle of the invention.

[0027] Figure 2a: TEM image of RBD nanoparticles used in the dissolving microneedle of the invention.

[0028] Figure 2b: Detail TEM image of RBD nanoparticles used in the dissolving microneedle of the invention. Figure 3: Graphical view of the cytotoxicity results of RBD-loaded nanoparticles used in the inventive dissolving microneedle.

[0029] Figure 4: Graphical view of the nitric oxide determination results of RBD-loaded nanoparticles used in the inventive dissolving microneedle.

[0030] Figure 5a: Optical microscope image of the inventive dissolving microneedles.

[0031] Figure 5b: Detail optical microscope image of the inventive dissolving microneedles.

[0032] Figure 6: View of the force-displacement graph of the inventive dissolving microneedles.

[0033] Figure 7a: SEM image of the inventive dissolving microneedles before force application.

[0034] Figure 7b: Detailed SEM image of the inventive dissolving microneedles before force application.

[0035] Figure 8a: SEM image of the inventive dissolving microneedles after force application.

[0036] Figure 8b: Detailed SEM image of the inventive dissolved microneedles after force application.

[0037] The invention relates to a dissolving microneedle, comprising nanoparticles loaded with receptor-binding domain (RBD).

[0038] Within the scope of the dissolving microneedle of the invention, RBD protein, which enables SARS-CoV-2 to bind with ACE2 receptors in the body, was selected as antigen. It is aimed to prepare a nanoparticle formulation to protect RBD from degradation in the body and to prolong its stay in the body, as well as to prevent repeated doses. Transdermal delivery of these nanoparticles through microneedles was aimed to increase the stability of the vaccine and to prevent cold chain application and fear of injection. It was aimed to develop an alternative vaccination strategy to traditional vaccine applications, to investigate the effectiveness of this application and to contribute against pandemics expected to be seen in the future.

[0039] As a result of formulation studies with different antigen amounts, internal and external phase ratios, sonication speed and duration, the optimum formula was obtained. In the selection of this formulation, the size of the RBD should be below 200 nm in order to be effectively taken up by antigen presenting cells (APCs) and the formulation in which the RBD is relatively more loaded was selected. For the microneedle formulation, parameters such as polymer concentration, centrifugation time and speed, drying temperature and time were varied and the formulation with the strength and smooth morphology that can pierce the skin was selected.

[0040] Production and characterization of RBD-loaded nanoparticles:

[0041] Preparation of nanoparticles;

[0042] Nanoparticles were prepared using solvent evaporation method. Firstly, 20 mg of poly(ethylene glycol)-poly(s-caprolactone) (PEG-PCL) polymer used in the study was dissolved in chloroform, the active substance solution was added and sonicated on ice at 30% power for 1 minute. This mixture was then transferred by syringe into a vial containing 1% sodium cholate and sonicated on ice at 30% power for 2 minutes. The resulting water / oil emulsion was transferred by syringe into a beaker containing 0.3% sodium cholate mixed at 600 rpm on a magnetic stirrer. The final formulation was mixed at 600 rpm for 3 hours. The water / oil / water emulsion was then centrifuged at 35000 rpm at 5 °C for 1 hour. The supernatant obtained at the end of centrifugation was stored at +4 °C for use in encapsulation efficiency and quantification. The precipitate obtained was used in nanoparticle characterization studies.

[0043] Characterization of nanoparticles:

[0044] Particle size (hydrodynamic diameter) and surface charge were analyzed by Dynamic Light Scattering (DLS) method using Zetasizer Nano ZS device. Analyzes performed in 3 series are expressed as average (Figure-1).

[0045] Quantification of the active substance in nanoparticle formulations was calculated by indirect method by determining the amount of active substance remaining in the supernatant without being loaded into the nanoparticle. For this purpose, the formulation was centrifuged for one hour at 35000 rpm using an ultra centrifuge. RBD encapsulation efficiency was calculated using the following equation

[0046] [(Amount of RBD loaded into nanoparticles - Amount of RBD in the supernatant) / amount of RBD initially used] x 100]

[0047] The average hydrodynamic diameter of RBD loaded nanoparticles was 192.8±0.3215 nm and the poly dispersity index (PDI) distribution was 0.331±0.058. The zeta potential was found to be -17.2±1.349.

[0048] The encapsulation efficiency was 95.583±1.107% for the RBD loaded nanoparticle.

[0049] The morphology and size of the particles were evaluated by Transmission Electron Microscopy at 120 kV.

[0050] When TEM images of the nanoparticles are examined, it is observed that spherical nanoparticles with smooth surfaces were obtained. In addition, it was found that the particle size observed by TEM in all formulas was similar to the average particle size measured by photon correlation spectroscopy method and in this respect, these two data support each other. Based on these images, it is proved that nanoparticles were successfully prepared (Figure-2).

[0051] Since the hydrodynamic diameter values of the nanoparticles are below 200 nm, they are considered to be suitable candidates for efficient uptake by antigen- presenting cells.

[0052] Cytotoxicity determination of nanoparticles:

[0053] The cytotoxicity of the nanoparticles was determined using the L929 cell line derived from fibroblasts. The effect of different concentrations of RBD-loaded nanoparticles, empty nanoparticles, PEG-PCL polymer and pure RBD on cell viability in L929 cell line was tested by a method called MTT. MTT assay is recognized as the gold standard for cell viability tests.

[0010]

[0054] For MTT assay, a 96-well plate was seeded with IxlO4cells / ml in each well and left to incubate for 24 hours. Selected concentration ranges were seeded on these cells. To determine the viability of the cells, 10 pl of 10 mg / ml MTT solution was added to each well after 72 hours of incubation. After 4 hours of incubation, 100 pl DMSO was added to each well to dissolve the formazan crystals that would form in the viable cells. After 30 min in the dark at room temperature, the optical density value in each well of the microplates was read at 540 nm on an ELISA Reader. At the end of 72 hours, the highest concentration that was non-toxic on cells was determined as 4 mg / ml. The results obtained are shown in Figure 3.

[0055] Nitric Oxide Determination:

[0056] Nitric oxide (NO) stimulates the production of cytokines in the body and promotes the destruction of intracellular pathogens via macrophages and enhances the immune response to infections. For this reason, NO is often used in vaccine studies. An increase in NO levels following interaction with antigen is an indication that an immune response has occurred.

[0011]

[0057] NO assay was performed using the J774 mouse macrophage cell line with a nontoxic concentration of RBD antigen and antigen-loaded nanoparticle formulation determined after cell viability analysis. 96-well microplates were coated with J774 macrophage cell lines and 50 pl of the sample at 3 different concentrations was added to the wells. The microplates were incubated at 37 °C for 72 hours, after which the supernatants in the wells were added to a new plate. 50 pl each of the standards prepared for analysis were also added to the wells. 50 pl of Griess reagent was added to each well. The microplate was kept in the dark for 10 min and UV measurement was performed at 540 nm. The amount of NO production was calculated for each sample by constructing a standard calibration curve. The results obtained are shown in Figure 4.

[0058] After 72 hours of incubation, it was determined that the nitric oxide levels produced by macrophages showed a 2.5-fold increase compared to the control group. The increase in nitric oxide levels over time indicates that antigen release occurs in a controlled manner from the nanoparticulate system and that the vaccine formulation has an immunostimulant effect.

[0059] Production and characterization of microneedles:

[0060] Production of microneedles:

[0061] Micro-molding method was used for the production of dissolving microneedles. Polymer solutions were applied to the polydimethylsiloxane (PDMS) mold and centrifuged. After drying in a desiccator, microneedles were removed from the mold using double-sided tape. Characterization of Microneedles:

[0062] Optical microscope images show that the microneedles have a uniform morphology. The images obtained are given in Figure 5.

[0063] TA.XT Plus texture analyzer was used to determine the mechanical strength of microneedles. The analysis was performed with a 10 mm diameter cylinder P / 10 probe. To test the strength of the microneedles, the microneedle array was attached to the probe using a double-sided tape with the needles facing downwards. The texture analyzer was set to compression mode. The P / 10 probe was moved downward at a speed of 1 mm / s to reach the metal surface with a trigger force of 0.049 N.

[0012]

[0064] In addition, the same analysis was repeated using parafilm cut into eight layers to simulate skin tissue. The parafilm layer was fixed on the metal surface using double-sided tape and the analysis was repeated by operating the device with the same operating principle.

[0013]

[0065] Both analyses were repeated with the TA.XT Plus device with both 5 kg and 30 kg load cells. Based on the data obtained, a force-displacement graph was created and the durability of microneedles was evaluated.

[0066] In order to determine the mechanical strength of the microneedles, force was applied with the texture analysis device and the results obtained were given as force-displacement graph. As a result of the analysis, a fracture peak could not be obtained in microneedles. The graph obtained is given in Figure 6. As can be seen from the graph, the microneedles have sufficient strength to pierce the skin tissue. Optical microscope results also showed that the microneedles did not break but bent. Microneedles were imaged with Scanning Electron Microscope before and after force application in order to see more clearly that the needles were not broken but bent. SEM images before force application are shown in Figure 7 and SEM images after force application are shown in Figure 8.

[0067] For the first time so far, it has been observed that the inventive dissolving microneedles can be used in vaccine applications and can generate immune responses. With the inventive dissolving microneedle, the RBD antigen will be encapsulated in the nanoparticle structure and will remain stable in the in vivo environment for a longer time. In this way, an extended immune response can be achieved. In addition, the absence of pain during vaccine administration greatly increases patient compliance. The invention, which is thought to greatly increase patient compliance and vaccination rate against the COVID-19 pandemic we have recently experienced and the pandemics and infections expected to be seen in the future, has the potential to be used as a result of further studies.

[0068] References:

[0069] [1] D. Sanyal, S. Chowdhury, V. Uversky, and K. Chattopadhyay, "An exploration of the SARS-CoV-2 spike receptor binding domain (RBD) - a complex palette of evolutionary and structural features," bioRxiv, p. 2020.05.31.126615, 2020, [Online], Available: https: / / doi.org / 10.1101 / 2020.05.31.126615.

[0070] [2] J. Lan et al., "Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor," Nature, vol. 581, no. 7807, pp. 215-220, 2020, doi : 10.1038 / s41586-020-2180-5. [3] F. Bayani et al., "An overview of the vaccine platforms to combat COVID- 19 with a focus on the subunit vaccines," Prog. Biophys. Mol. BioL, vol. 178, no. September 2022, pp. 32-49, 2023, doi: 10.1016 / j.pbiomolbio.2023.02.004.

[0071] [4] P. S. Arunachalam et al., "Adjuvanting a subunit COVID-19 vaccine to induce protective immunity," Nature, vol. 594, no. 7862, pp. 253—258, 2021, doi: 10.1038 / s41586-021-03530-2.

[0072] [5] G. Rodrigues, M. Goncalves Da Costa Sousa. D. C. Da Silva. T. M. Berto Rezende, P. C.De Morais. and O. L. Franco, "Nanostrategies to Develop Current Antiviral Vaccines." ACS Appl. Bio Mater., vol. 4, no. 5, pp. 3880-3890, 2021, doi: 10.1021 / acsabm.0c01284.

[0073] [6] C. Feng et al., "Emerging vaccine nanotechnology: From defense against infection to sniping cancer," Acta Pharm. Sin. B, vol. 12, no. 5, pp. 2206-2223, 2022, doi: 10.1016 / j.apsb.2021.12.021.

[0074] [7] M. S. Lee, C. X. Pan, and V. E. Nambudiri, "Transdermal approaches to vaccinations in the COVID-19 pandemic era," Ther. Adv. Vaccines Immunother., vol. 9, pp. 1-13, 2021, doi: 10.1177 / 25151355211039073.

[0075] [8] S. M. Kapnick. "The Nanoparticle-Enabled Success of COVID-19 mRNA Vaccines and the Promise of Microneedle Platforms tor Pandemic Vaccine Response," DNA Cell Biol., vol. 41, no. 1, pp. 25-29, 2021, doi: 10.1089 / dna.2021.0538.

[0076] [9] L. K. Vora et al., "Microneedle array systems for long-acting drug delivery," Eur. J. Pharm. Biopharm., vol. 159, no. December 2020, pp. 44-76, 2021, doi: 10.1016 / j.ejpb.2020.12.006.

[0010] Pinar Erkekoglu and Terken Baydar, "Gtincel in Vitro Sitotoksisite Testi eri,” Hacettepe Univ. J. Fac. Pharm., vol. 41, no. 1, pp. 45-63, 2021.

[0077]

[0011] S. Derman, Z. A. Mustafaeva, E. S. Abamor, M. Bagirova, and A. Allahverdiyev, "Preparation, characterization and immunological evaluation: Canine parvovirus synthetic peptide loaded plga nanoparticles," J. Biomed. Sci., vol. 22, no. 1, pp. 1-12, 2015, doi: 10.1186 / sl2929-015-0195-2.

[0078]

[0012] A. Panda, P. K. Sharma, T. McCann, J. Bloomekatz, M. A. Repka, and S. N. Murthy, "Fabrication and development of controlled release PLGA microneedles for macromolecular delivery using FITC-Dextran as model molecule," J. Drug Deliv. Sci. Technol., vol. 68, no. November 2020, p. 102712, 2022, doi: 10.1016 / j.jddst.2021.102712.

[0079]

[0013] A. J. Paredes et al., "Ring inserts as a useful strategy to prepare tip-loaded microneedles for long-acting drug delivery with application in HIV pre-exposure prophylaxis," Mater. Des., vol. 224, p. 111416, 2022, doi: 10.1016 / j.matdes.2022.111416.

Claims

CLAIMS1. The invention relates to a dissolving microneedle, characterized in that it comprises nanoparticles loaded with a receptor-binding domain (RBD).

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