Hybrid nanocarrier system
The hybrid nanocarrier system addresses toxicity and stability issues by using flavonoids for green-chemistry reduction and pH-responsive release, enabling dual therapy and efficient delivery of chemotherapeutic and gene agents, with rapid purification and Raman tracking.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- ERCİYES ÜNİVERSİTESİ STRATEJİ GELİŞTİRME DAİRE BAŞKANLIĞI
- Filing Date
- 2025-10-22
- Publication Date
- 2026-04-30
AI Technical Summary
Existing metal nanoparticle-based carrier systems face issues such as toxicity, stability, immune response triggers, prolonged purification times, and inability to carry both chemical and genetic materials simultaneously, with low drug delivery efficiency and layer binding/adhesion.
A hybrid nanocarrier system with a metallic core and biocompatible outer layers, using flavonoids for green-chemistry reduction and pH-responsive release, enabling dual therapy modalities and Raman tracking, with strong electrostatic binding and rapid layer integration.
The system provides stable, biocompatible, and efficient delivery of both chemotherapeutic and gene therapy agents, preventing toxicity and immune responses, while allowing rapid purification and synergistic cancer treatment.
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Abstract
Description
[0001] HYBRID NANOCARRIER SYSTEM
[0002] FIELD OF THE INVENTION
[0003] The present invention relates to a hybrid nanocarrier system designed for use in fields such as gene therapy, cancer treatment, chemotherapy, the treatment of genetic diseases, innovative vaccine technologies, the production of innovative antibiotics for antibacterial and antimicrobial therapies, drug delivery, the dietary supplement market, Raman imaging systems, biotechnological applications, and the agrochemical industry.
[0004] BACKGROUND OF THE INVENTION
[0005] In carrier systems formed using metal nanoparticles in the state of the art, the occurrence of toxicity issues is quite likely. Some metallic nanomaterials may be toxic in the body and may lead to long-term biological effects. In particular, nanoscale particles can interact with cells and cause undesired effects.
[0006] Inorganic reducing agents used in the production of metal nanomaterials can exhibit toxicity to organisms when the purification steps are not properly performed.
[0007] Metal nanomaterials can trigger immune responses when they enter the body. This may lead to side effects such as allergic reactions or inflammation.
[0008] Metal nanomaterials may exhibit high reactivity and may suffer from stability problems. This, in turn, can affect drug delivery and release and reduce therapeutic efficacy. The production of carrier systems prepared using the layer-by-layer (LbL) method available on the market is generally quite time-consuming due to long incubation times and prolonged purification steps.
[0009] In layered systems, the homogeneous incorporation of the target compounds into the systems and the control of their release present problems for the carrier systems on the market. In addition, the efficiencies of drug delivery and of layer binding / adhesion are low.
[0010] None of the carrier systems on the market is capable of carrying both a chemical material and, at the same time, genetic material.
[0011] In the literature search, U.S. document US10278927B2 relating to particle coating was identified. In the invention in question, the particles are coated with poly-L-arginine (PLA) in a first layer, with a siRNA therapeutic agent added in a second layer, with PLA in a third layer, and with hyaluronic acid (HA) in a final layer.
[0012] Another document in the literature on the subject, US9393217B2, discusses the process steps for protein and drug delivery applications. The layers are as follows: a degradable polyelectrolyte layer having a first electrostatic charge; a first polysaccharide layer disposed adjacent to the degradable poly electrolyte layer; a carrier layer disposed adjacent to the first polysaccharide layer; and a second polysaccharide layer disposed adjacent to the carrier layer.
[0013] DESCRIPTION OF THE INVENTION
[0014] In the developed system, the outermost layer is coated with a polymer having very high biocompatibility, thereby preventing all potential toxic effects. Since the metal nanoparticles are located in the core portion of the system, they will not directly interact with cells, and problems such as triggering the immune system during circulation will not arise.
[0015] The designed particles have a metallic core and, due to the presence of the overlying layers, a certain mass. Thanks to this mass, they can readily be collected as a pellet by centrifugation for storage and can maintain their stability for extended periods.
[0016] The electrostatic attraction between the layers of the invention is strong due to the optimal adjustment of their differences in surface charge. Accordingly, the incubation times required for each layer to be integrated into the system are much shorter than those of systems on the market. In addition, owing to the molecular weight of the system, it can be rapidly centrifuged and easily purified.
[0017] Because the designed particles are pH-responsive, they can fully release all of their cargo into the cell at the endosomal stage.
[0018] The metal nanoparticles within the system are reduced by flavonoids — a class of natural compounds — using green-chemistry methods. In this way, the formation of any toxic effects due to residues of inorganic reducing agents is prevented. In addition, when the flavonoids are introduced into the system in a basic solution such as NaOH, the metal nanoparticles are observed to possess a much more negative surface charge, thereby enabling much stronger electrostatic binding of the subsequent layer to the system.
[0019] The present invention enables the simultaneous use of dual therapy modalities by both retaining a chemical compound of a flavonoid type around the metallic core and arranging any desired genetic material (mRNA, siRNA, miRNA, plasmid, etc.) on the cationic (positive) layer. At the same time, owing to the presence of Raman-active molecules (4- ATP, MBA, MPY, PYOT, etc.), the invention can be tracked by Raman spectroscopy within cells and in tissue environments. Thus, it also confers a theranostic capability.
[0020] One of the most important features of the present invention is that, compared with counterparts, it can deliver both chemotherapeutic agents and gene therapy agents. Accordingly, innovative cancer vaccines can be developed and — depending on the flavonoid and the genetic agent employed — antibiotic resistance can be prevented.
[0021] Another feature of the invention is that any desired metal nanoparticle, any desired flavonoid, and any desired genetic material can be readily integrated into the system, thereby enriching its range of applications and allowing classification according to need.
[0022] Another feature of the invention is that, owing to the applied technique, it has been observed that flavonoids dissolved in a basic solution yield nanoparticles with a much more negative charge, whereby the integration of layers onto one another becomes both much faster and much stronger than in counterparts.
[0023] Description of Drawings
[0024] Figure 1. Depiction of the formation process of the hybrid nanoparticles planned to be produced by the layer-by-layer (LbL) method.
[0025] Figure 2. Schematic of the layers of the hybrid nanoparticle (HNP) and the positions of the molecules contained therein.
[0026] Figure 3. Synthesis process of the hybrid nanoparticles planned to be produced by the LbL method.
[0027] Figure 4a. Particle size measurement results obtained by Nanoparticle Tracking Analysis (NTA) after the addition of each layer to the HNPs.
[0028] Figure 4b. Surface charge (zeta potential) measurement results obtained by a Zetasizer instrument after the addition of each layer to the HNPs. Figure 5. STEM micrograph(s) of different layers of the HNPs.
[0029] Figure 6a. Resazurin (alamarBlue) cell viability assay results for MDA-MB-231, BT-549, and 4T1 cells treated with: blank HNP; HNP loaded only with eEF2K-siRNA (siRNA-HNP); HNP loaded only with quercetin (QU-HNP); and HNP co-loaded with eEF2K-siRNA and quercetin (siRNA-QU-HNP) at different concentrations.
[0030] Figure 6b. Colony formation (clonogenic) assay images for MDA-MB-231, BT-549, and 4T1 cells treated with different HNP concentrations, with doxorubicin (DOX) as a positive control.
[0031] Figure 6c. Quantitative results from ImageJ for the colony formation assay in the MDA-MB-231 cell line (colonies defined as clusters of more than 50 cells).
[0032] Figure 6d. Quantitative results from ImageJ for the colony formation assay in the BT-549 cell line (colonies defined as clusters of more than 50 cells).
[0033] Figure 6e. Quantitative results from ImageJ for the colony formation assay in the 4T1 cell line (colonies defined as clusters of more than 50 cells).
[0034] Figure 7a. Wound-healing (scratch) assay images for MDA-MB-231, BT-549, and 4T1 cells after treatment with HNPs at different concentrations.
[0035] Figure 7b. Quantitative wound-gap distance results (pm scale) for the MDA-MB-231 cell line (computed in ImageJ and analyzed in GraphPad).
[0036] Figure 7c. Quantitative wound-gap distance results (pm scale) for the BT-549 cell line (computed in ImageJ and analyzed in GraphPad).
[0037] Figure 7d. Quantitative wound-gap distance results (pm scale) for the 4T1 cell line (computed in ImageJ and analyzed in GraphPad).
[0038] Figure 8a. Apoptosis assay results for MDA-MB-231, BT-549, and 4T1 cells treated with HNPs at different concentrations (Annexin V and propidium iodide (PI) staining).
[0039] Figure 8b. Quantitative results for necrotic levels in MDA-MB-231 cells.
[0040] Figure 8c. Quantitative results for necrotic levels in BT-549 cells.
[0041] Figure 8d. Quantitative results for necrotic levels in 4T1 cells.
[0042] Figure 9. Seven-day evaluation results for 3D tumor spheroid models of MDA-MB-231 cells treated at different HNP concentrations. Figure 10. Seven-day evaluation results for 3D tumor spheroid models of BT-549 cells treated at different HNP concentrations.
[0043] Figure 11. Seven-day evaluation results for 3D tumor spheroid models of 4T1 cells treated at different HNP concentrations.
[0044] Figure 12a. Synthesis scheme for the conjugation of HNPs with the Raman-active molecule 4-aminothiophenol (4-ATP).
[0045] Figure 12b. Raman spectra acquired from each layer of the HNP architecture.
[0046] Figure 12c. Raman imaging of cells treated with HNPs and spectra obtained from these images (scale bar: 20 pm for the Raman image; 50 pm for the bright-field image).
[0047] DETAILED DESCRIPTION OF THE INVENTION
[0048] In this detailed description, the hybrid nanocarrier system that is the subject of the invention, together with its preferred alternatives, is described solely to facilitate understanding of the subject and is not intended to be limiting in any way.
[0049] 1. MeNP+Fl synthesis:
[0050] First, preferably 10 mM of a metal salt (gold, silver, copper, iron, platinum, titanium, etc.) is prepared and added into a vial, then preferably 2 mM of the flavonoid (Fl) is added dropwise with stirring. At room temperature and under stirring, the volume is brought to 5 mL with dFbO, after which the mixture is incubated for 2 hours. At the end of this stage, metal nanoparticles (MeNPs) are obtained.
[0051] Subsequently, the MeNP+Fl particles are washed with dlLO. First, the nanoparticles are pelleted preferably at 15,000 rpm for 20 minutes, the supernatant is removed, and the pellet is redispersed in dH2O. Then, the particles are centrifuged again at 15,000 rpm for 20 minutes, thereby completing the washing step.
[0052] 2. MeNP+Fl+RAM synthesis: 30 mM of the Raman-active molecule (RAM) is weighed and dissolved in 1 mL of dJLO. It is then mixed with 1 mL of the previously synthesized MeNP+Fl. The total volume is brought to 5 mL with dH>0 and the mixture is incubated at 65 °C for 12 h on a magnetic stirrer, after which washing is performed as in the MeNP+Fl synthesis. In this way, conjugation via the -SH groups of the Raman-active molecules forms a thiolate-metal (S-M) bond with the metal nanoparticles, thereby completing and purifying the synthesis.
[0053] 3. MeNP+Fl+RAM+PAH synthesis:
[0054] 41 mM of poly(allylamine hydrochloride) (PAH) is weighed and dissolved in 1 mL of dH>0. Then, it is mixed with 667 pL of MeNP+Fl+RAM, the total volume is brought to 5 mL with dH>0, and the mixture is incubated at room temperature on a magnetic stirrer for 3 h. Finally, washing is performed as in the MeNP+Fl synthesis. At the end of this step, the outermost part of the system is coated with PAH, a positively charged polymer, thereby preparing the system for genetic material loading.
[0055] 4. MeNP+Fl+RAM+PAH+GM synthesis:
[0056] From the product prepared in the previous step, 227 pL is taken and mixed with 1.25 pL of 100 pM genetic material (GM), and the mixture is brought to a total volume of 500 pL. The system is then incubated on an orbital shaker at room temperature for 1 h. In this way, owing to the negative charges of the phosphate groups of the genetic materials and the positively charged shell provided by the PAH layer, the particle achieves electrostatic association (complexation) with the genetic material.
[0057] 5. MeNP+Fl+RAM+PAH+GM+PSS synthesis:
[0058] 36 mM of poly (styrene sulfonate) (PSS) is weighed and dissolved in 1 mL of dH>0. It is mixed with the entirety of the 500 pL of particles synthesized in the preceding step, and the total volume is brought to 5 mL. Thereafter, the mixture is incubated on a magnetic stirrer at room temperature for 3 h, and washing is performed as in the MeNP+Fl synthesis. Following this step, the negatively charged PSS molecules electrostatically bind to the positively charged surface arising from PAH, and the particle is rendered fully biocompatible. The invention is designed to be widely applicable in cancer research and therapy. Accordingly, in an exemplary scenario, a researcher aiming to treat triple-negative breast cancer (TNBC) may produce silver nanoparticles using the flavonoid quercetin, which is frequently employed in chemotherapeutic applications, and — after adding the other layers of our system as described — may integrate into the system a siRNA molecule that suppresses the eEF2K gene, a potential target for breast cancer. In this way, the invention can readily be employed as a cancer vaccine with a synergistic effect, by combining both chemotherapy and gene therapy modalities.
Claims
CLAIMS1. A hybrid nanocarrier system applicable in many fields, primarily cancer therapy, gene therapy, and antibacterial treatments, the system being characterized by comprising the following process steps:- reducing metal salts to metal nanoparticles using a flavonoid;- dissolving the flavonoids in an NaOH solution upon their addition to the system, thereby imparting a more negative charge to the core of the carrier system;- covalently integrating Raman-active molecules into the metallic nanoparticle core; - coating the metal core with poly(allylamine hydrochloride), a positively charged polymer;- incorporating desired genetic material into the system via electrostatic forces;- coating with negatively charged, highly biocompatible poly(styrene sulfonate) to increase biocompatibility and protect the incorporated genetic material.
2. The invention according to claim 1 , being a hybrid nanocarrier system, characterized in that, to enable the binding of Raman-active molecules to the metal nanoparticles, it comprises the steps of weighing preferably 30 mM of a Raman-active molecule and dissolving it in preferably 1 mL of dJLO; mixing it with 1 mL of the previously synthesized MeNP+Fl; adding dJLO to bring the total volume to 5 mL; incubating preferably at 65 °C for 12 hours; and washing.
3. The invention according to claim 1 , being a hybrid nanocarrier system, characterized in that the coating of the metal core with poly(allylamine hydrochloride) (PAH) comprises the steps of weighing preferably 41 mM of PAH and dissolving it in 1 mL of dH>0; mixing with 667 pL of MeNP+Fl+RAM and bringing the total volume to 5 mL with dH>0; incubating at room temperature on a magnetic stirrer for about 3 hours; washing as in the MeNP+Fl synthesis; and coating the outermost part of the system with PAH.
4. The invention according to claim 1 , being a hybrid nanocarrier system, characterized in that, in order to enable incorporation of the desired genetic material by electrostatic forces, it comprises the steps of taking 227 pL of the MeNP+Fl+RAM+PAH product and mixing it with 1.25 pL of 100 pM genetic material (GM); bringing the total volume to 500 pL; and incubating the system on an orbital shaker at room temperature, preferably for 1 hour.
5. The invention according to claim 1 , being a hybrid nanocarrier system, characterized in that, in order to increase biocompatibility and protect the incorporated genetic material, it comprises the steps of dissolving 36 mM of poly(styrene sulfonate) (PSS) in 1 mL of dJLO; mixing with the entirety of the 500 pL of MeNP+Fl+RAM+PAH+GM particles; bringing the total volume to 5 mL; incubating at room temperature for 3 hours; and washing.