Stable polymer lipid nanoparticles for binding and delivery of drugs, particularly drugs having anticancer properties, and methods for producing stable polymer lipid nanoparticles for transporting drugs, particularly drugs having anticancer properties.

Abstract

The present invention discloses stable polymer lipid nanoparticles for drug binding and delivery, comprising a core containing a polylactic acid-glycolic acid copolymer (PLGA) and polyvinyl alcohol (PVA), and a core envelope containing a lipid mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DDPC), cholesterol, and an ammonium salt of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000(DSPE-PEG(2000)NH2)], as well as a method for producing the same.

Description

[Technical Field]

[0001] The present invention relates to stable polymer lipid nanoparticles for the binding and delivery of drugs, particularly drugs having anticancer properties, and to methods for producing stable polymer lipid nanoparticles that support drugs, particularly drugs having anticancer properties. [Background technology]

[0002] Polylactic acid-glycolic acid copolymer (PLGA) is an FDA-approved copolymer of lactic acid and glycolic acid. It is also one of the most widely used polymers in the design and formulation of drug delivery systems for biomedical applications due to its biodegradability, biosafety, biocompatibility, formulation versatility, and functionalization. PLGA-based nanoparticles provide optimal bioavailability of encapsulated drugs, protection from premature degradation of encapsulated drugs in the biological environment, regulation of degradation rates, and targeted drug delivery. Furthermore, PLGA-based nanoparticles facilitate intracellular penetration of bioactive compounds and reduce therapeutic side effects. PLGA nanoparticles are widely used in the following treatments: anti-cancer [DOI:10.1615 / CritRevTherDrugCarrierSyst.v21.i5.20], anti-inflammatory [https: / / doi.org / 10 / 1039 / C5RA17686G], cardiovascular and immunological [https: / / doi.org / 10.1016 / j.drudis.2014.09.018,https: / / doi.org / 10.1039 / C5TB00434A].

[0003] The use of PLGA allows for the encapsulation of a wide range of bioactive molecules, including small drugs, proteins, and nucleic acids. Due to its low intrinsic toxicity and easy biodegradability, PLGA-containing nanoparticles can be used in formulations for systemic (parenteral), oral, and inhalation administration.

[0004] Nevertheless, despite the attractive characteristics of PLGA-based nanoparticles, there are several limitations typically associated with their physicochemical and biological properties, among which we can mention low drug saturation (especially in the case of lipid-soluble therapeutics), drug release in the first few hours of administration, phagocytic uptake, short half-life, aggregation, immune response, and uncontrolled distribution in tissues [https: / / doi.org / 10.1016 / j.drudis.2014.09.018,https: / / doi.org / 10.1080 / 17425247.2016.1182492]. These properties limit the use of PLGA nanoparticles in biomedicine, and therefore, hybrid nanoparticles in which PLGA is combined with protective materials, primarily polymers, surfactants, and lipids, are being explored.

[0005] Polymer PVA, or poly(vinyl alcohol), forms particles with a relatively small size and uniform size distribution, making it the most commonly used emulsifier to stabilize emulsification during the formulation of PLGA nanoparticles. Because PVA forms an interconnected network with the polymer at the interface, the PVA coating remains bound to the nanoparticles despite repeated washing [https: / / doi.org / 10.1016 / 0168-3659(95)00070-O]. PVA coating can alter the properties of PLGA nanoparticles and reduce intracellular uptake of nanoparticles, which is associated with increased hydrophilicity of the nanoparticle surface and negative charges on the nanoparticle surface.

[0006] PLGA lipid hybrid nanoparticles are becoming an increasingly promising approach in modern nanomedicine, minimizing side effects while improving pharmacokinetics and the biodistribution of therapeutic systems in the biological environment. PLGA lipids combine the characteristics of biomimetic lipids with the structural advantages of PLGA nanoparticles into a single entity [https: / / doi.org / 10.1517 / 17425247.2016.1151872]. Among polymer lipid materials, we distinguish between lipid-core polymer envelopes and polymer-core lipid envelope nanoparticles. A second type of hybrid nanostructure is a biodegradable PLGA core surrounding a therapeutic agent, consisting of a biodegradable PLGA core embedded in a polyethylene glycol (PEG)-modified lipid or lipid envelope [https: / / doi.org / 10.2147 / IJN.S40579,https: / / doi.org / 10.1039 / C2NR32880A,https: / / doi.org / 10.1016 / j.ejpb.2013.07.002]. The outer lipid layer reduces the degradation rate of the PLGA core, limits the diffusion of water within the molecule, and enables more controlled release kinetics. Furthermore, this layer acts as a molecular barrier that minimizes drug leakage during preparation or storage [https: / / doi.org / 10.1039 / C2NR32880A,https: / / doi.org / 10.1016 / j.ejpb.2013.07.002]. The outer layer of the carrier is also involved in the carrier's circulation time in the bloodstream and its accumulation in specific organs and tissues. Therefore, the composition of the outer lipid layer is a crucial factor in the design of polymer lipid drug carriers, giving them unique characteristics. [Overview of the project]

[0007] The essence of the solution according to the present invention relating to polymer lipid carriers is that the carrier is composed of a core envelope containing a lipid mixture comprising a core containing 16.1 to 30.3% by weight of polylactic acid-glycolic acid copolymer (PLGA) and 60.6 to 75.5% by weight of polyvinyl alcohol (PVA), and an ammonium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DDPC) in the range of 3.66% to 12.6% by weight, cholesterol in the range of 1.85 to 6.29% by weight, and an ammonium salt of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000(DSPE-PEG(2000)NH2)] in the range of 0.15 to 0.5% by weight.

[0008] Advantageously, PLGA is a 50:50 copolymer with Mw of 24,000 to 38,000 g / mol. Advantageously, PVA has a mass Mw of 30,000 to 70,000 g / mol.

[0009] The essence of the solution provided by the present invention, relating to the method, In the first step, an aqueous PVA solution with a concentration of 15 mg / ml to 20 mg / ml is prepared, followed by a PLGA dichloromethane solution with a concentration of 10 mg / ml to 20 mg / ml. Then, at least one drug is weighed in the amount necessary to prepare a solution with a concentration in the range of 0.43 mM to 4.30 mM and dissolved in the PLGA solution. After that, both solutions are cooled to 4°C to 7°C and then mixed in a volume ratio of 1:2 (equal parts of 2 parts aqueous PVA solution to 1 part PLGA dichloromethane solution containing the drug). The mixture is then cooled again to 4°C to 7°C, mixed to form an emulsion, poured into a crystallizer with a stirring element, the residue is washed with PBS buffer pH 7.4 or water in a volume ratio of rinse solution (PBS or water) to reaction mixture of 1:7.5 to 1:10, and this is also placed into the crystallizer, then the dichloromethane is completely evaporated, and then the core is obtained by replenishing the volume with PBS 7.4 solution or water until a final PLGA concentration of 3.33 mg / ml to 6.66 mg / ml is obtained. In the second step, a core pre-formed with DPPC / cholesterol / DSPE-PEG(2000)NH2 liposomes (1.0 / 0.61 / 0.040) synthesized by the selected method is covered, the formed lipid membrane is hydrated with 0.9% NaCl sterile saline until the total lipid concentration reaches 10 mg / ml, and dispersed by sonication to obtain a DPPC / cholesterol / DSPE-PEG(2000)NH2 liposome solution as an intermediate for further synthesis. Then, the obtained liposomes are added to an unpurified PVA / PLGA polymer core in a volume ratio of liposomes to polymer core of 0.1:1 to 0.3:1, and the whole is then heated at 45 to 60°C for 2 to 4 hours with stirring at 200 to 600 RPM. The resulting PLGA / PVA / LIPO nanoparticles are left at 4°C for 12 to 24 hours and purified by washing twice with PBS buffer pH 7.4.

[0010] Advantageously, PLGA is a 50:50 copolymer with Mw of 24,000 to 38,000 g / mol.

[0011] Advantageously, PVA has a mass Mw of 30,000 to 70,000 g / mol. Advantageously, 0.1 M PBS buffer pH 7.4 is added to the PVA solution. Advantageously, the internal core polymer is a poly(L-lactic acid-glycolide) copolymer (PLGA).

[0012] Advantageously, the outer polymer of the core is poly(vinyl alcohol) PVA. Preferably, the drug introduced into the core is 5-11.5 mg of staurosporine.

[0013] Preferably, the drug introduced into the core is 5-11.5 mg of 17AAG (tanespimycin). Preferably, staurosporine and 17AAG (tanespimycin) are introduced into the core at a concentration ratio of 17AAG (tanespimycin) to staurosporine of 0.62 to 9.5.

[0014] Advantageously, the evaporation of dichloromethane is carried out at a temperature of 37°C to 40°C using a magnetic stirrer at a speed of 200 to 600 RPM. Advantageously, the emulsion is obtained by stirring with an ultrasonic probe at an amplitude of 70% to 90% for 1 to 2 minutes.

[0015] Advantageously, dynamic light scattering is used to measure the size of nanoparticles in order to control the quality of the fabricated PLGA / PVA polymer cores. Advantageously, DPPC / cholesterol / DSPE-PEG(2000)NH2 liposomes are synthesized by hydration using a chloroform solution of a lipid mixture in the ratio of 96.0 mM equivalent of DPPC, 58.8 mM equivalent of cholesterol, and 3.84 mM equivalent of DSPE-PEG(2000)NH2.

[0016] Advantageously, DPPC / cholesterol / DSPE-PEG(2000)NH2 liposomes are synthesized by ethanol injection by preparing an ethanol solution of a lipid mixture in the ratio of 96.0 mM equivalent of DPPC, 58.8 mM equivalent of cholesterol, and 3.84 mM equivalent of DSPE-PEG(2000)NH2.

[0017] Advantageously, in the hydration method, the solvent is evaporated using a vacuum evaporator or vacuum dryer. Advantageously, in the ethanol injection method, an ethanol solution of lipids is added dropwise to sterile physiological saline mixed with a magnetic stirrer to obtain a solution of DPPC / cholesterol / DSPE-PEG(2000)NH2 liposomes having a total lipid concentration of 10 mg / ml as an intermediate for further synthesis.

[0018] Advantageously, the purification of nanoparticles is performed by high-speed centrifugation at 15,000 RPM for 2–6 minutes. Advantageously, after each addition of PBS7.4, use ultrasound for at least 5 seconds to disperse the nanoparticles.

[0019] Advantageously, to stabilize the nanoparticles, after the last centrifugation, a 40% solution of a non-ionic surfactant or glucose with a volume ratio of 0.02:1 (surfactant:nanoparticles) is added after stirring the solution until a concentration of 2 mg / ml is achieved.

[0020] Advantageously, the drug content, i.e., %EE, is determined by UV-Vis spectrophotometry or LC-MS for single or mixed drugs by dissolving PLGA / PVA / LIPO nanoparticles in DMSO containing HCl according to the scheme: 25 - 75 μL of sample, 50 μL of 0.3 M HCl, 25 - 75 μL of PBS 7.4, and 850 μL of DMSO.

[0021] The main advantages of the solution according to the present invention are that the combination of the polymer and lipid used in the nanoparticles enables loading at levels of 32% - 80% of single drugs such as 17AAG (tanespimycin) and staurosporine, as well as combinations thereof; sustained release of the drug or drug combination, prevention of excessive drug leakage and aggregation of the carrier in the presence of plasma proteins. In addition, a concentration ratio range of 0.62 - 9.5 of 17AAG to staurosporine is possible. The lipid bilayer coating using polyethylene glycol enables prevention of nanoparticle aggregation in the presence of proteins and ensures the stability of the nanoparticles over 96 hours at 37°C. The size of the PLGA / PVA / LIPO nanoparticles in the range of 209 nm - 388 nm ensures selective passive transport of the drug to tumor tissue due to the effect of increased permeability and EPR retention. In addition, further modification of polyethylene glycol with amino groups gives the nanoparticles a low positive potential of 6.5 mV - 9.5 mV, enabling enhanced anti-tumor effects, better accumulation and penetration of the carrier in tumor tissue, and better cell uptake compared to inert negatively charged nanoparticles (https: / / doi.org / 10.1016 / j.nantod.2016.04.008). Such a developed structure results in the developed nanoparticles having a small diameter and a low PDI polydispersity index.

[0022] Briefly, the developed nanoparticles provide a high drug loading rate, reduced initial drug release and sustained drug release, a positive zeta potential for carrier accumulation in tumor tissue, and long-term stability of the nanoparticles over 96 hours in the presence of plasma proteins.

[0023] The solution according to the present invention will be described by the following production examples and drawings.

Brief Description of Drawings

[0024] [Figure 1.1] An example of the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / 1G nanoparticles measured by dynamic light scattering is shown. [Figure 1.2] An example of the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / 3G nanoparticles measured by dynamic light scattering is shown. [Figure 1.3] Figure 1.3A shows the UV-Vis spectrum of PLGA / PVA / LIPO / 17AAG / 3G nanoparticles dissolved in DMSO containing hydrochloric acid according to the ratio of 50 μL of PLGA / PVA / LIPO / 17AAG / 3G nanoparticles, 50 μL of 0.3 M HCl, 50 μL of PBS7.4, and 850 μL of DMSO. Figure 1.3B shows the calibration curve of staurosporine recorded in a solution of 50 μL of 0.3 M HCl, 100 μL of PBS7.4, and 850 μL of DMSO. [Figure 1.4] The dependence of the growth inhibition of 8MGBA cells (human glioma) on the concentration of free and encapsulated 17AAG in PLGA / PVA / LIPO / 17AAG / 3G nanoparticles is shown. [Figure 1.5] The dependence of the growth inhibition of A172 cells (human glioma) on the concentration of free and encapsulated 17AAG in PLGA / PVA / LIPO / 17AAG / 3G nanoparticles is shown. [Figure 1.6] The dependence of the growth inhibition of Hs683 cells (human glioma) on the concentration of free and encapsulated 17AAG in PLGA / PVA / LIPO / 17AAG / 3G nanoparticles is shown. [Figure 1.7] This study demonstrates the dependence of LN-229 cell (human brain glioma) proliferation inhibition on the concentration of free and encapsulated 17AAG in PLGA / PVA / LIPO / 17AAG / 3G nanoparticles. [Figure 1.8] This study demonstrates the dependence of LUDLU-1 cell (human brain glioma) proliferation inhibition on the concentration of free and encapsulated 17AAG in PLGA / PVA / LIPO / 17AAG / 3G nanoparticles. [Figure 1.9] This study demonstrates the dependence of NCl-H226 cell proliferation inhibition in human lung squamous cell carcinoma on the concentration of free and encapsulated 17AAG in PLGA / PVA / LIPO / 17AAG / 3G nanoparticles. [Figure 1.10] This study demonstrates the dependence of NCl-H520 cell proliferation inhibition in human lung squamous cell carcinoma on the concentration of free and encapsulated 17AAG in PLGA / PVA / LIPO / 17AAG / 3G nanoparticles. [Figure 1.11] This study demonstrates the dependence of inhibition of SK-MES-1 cell proliferation (human lung squamous cell carcinoma) on the concentration of free and encapsulated 17AAG in PLGA / PVA / LIPO / 17AAG / 3G nanoparticles. [Figure 2.1] An example of the zeta potential distribution of PLGA / PVA / LIPO / ST / 3G nanoparticles before surfactant addition, measured by dynamic light scattering, is shown. The sample was diluted 20-fold with distilled water. [Figure 2.2] This shows an example of the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / ST / 4T nanoparticles, measured by dynamic light scattering. [Figure 2.3] Figure 2.3A shows the UV-Vis spectra of PLGA / PVA / LIPO / ST / 4T nanoparticles dissolved in DMSO containing hydrochloric acid according to the ratio of 25 μL of PLGA / PVA / LIPO / ST / 4T nanoparticles, 50 μL of 0.3 M HCl, 75 μL of PBS7.4, and 850 μL of DMSO. Figure 2.3B shows the calibration curve of staurosporine recorded in a solution of 50 μL of 0.3 M HCl, 100 μL of PBS7.4, and 850 μL of DMSO. [Figure 2.4]This study demonstrates the dependence of A172 cell (human glioblastoma multiforme) proliferation inhibition on the concentration of free and PLGA / PVA / LIPO / ST / 4T nanoparticles. [Figure 2.5] This study demonstrates the dependence of A172 cell (human glioblastoma multiforme) proliferation inhibition on the concentration of free and PLGA / PVA / LIPO / ST / 4T nanoparticles. [Figure 2.6] This study demonstrates the dependence of Hs683 cell (human brain glioma) proliferation inhibition on the concentration of free and PLGA / PVA / LIPO / ST / 4T nanoparticles. [Figure 2.7] This study demonstrates the dependence of LN-229 cell (human brain glioma) proliferation inhibition on the concentration of free and PLGA / PVA / LIPO / ST / 4T nanoparticles. [Figure 2.8] This study demonstrates the dependence of LUDLU-1 cell (human brain glioma) proliferation inhibition on the concentration of free and PLGA / PVA / LIPO / ST / 4T nanoparticles. [Figure 2.9] This study demonstrates the dependence of NCl-H226 cell proliferation inhibition in human lung squamous cell carcinoma on the concentration of free and PLGA / PVA / LIPO / ST / 4T nanoparticles. [Figure 2.10] This study demonstrates the dependence of NCl-H520 cell proliferation inhibition in human lung squamous cell carcinoma on the concentration of free and PLGA / PVA / LIPO / ST / 4T nanoparticles. [Figure 2.11] This study demonstrates the dependence of inhibition of SKMES-1 cell proliferation (human lung squamous cell carcinoma) on the concentration of free and PLGA / PVA / LIPO / ST / 4T nanoparticles. [Figure 3.1A] This shows an example of the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles, measured by dynamic light scattering. [Figure 3.1B] An example of the zeta potential distribution of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles before surfactant addition, measured by dynamic light scattering, is shown. [Figure 3.2] This shows plots of the hydrodynamic diameter of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles against temperature, recorded 24 hours and 7 days after synthesis (for pH 7.4) in buffers with pH specific to endosomes and lysosomes (pH 5, pH 6) and the natural pH (pH 7.4, specific to blood flow). [Figure 3.3] This shows temperature-dependent plots of the PDI polydispersity index of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles, recorded 24 hours and 7 days after synthesis (for pH 7.4) in buffers with pH values ​​specific to endosomes and lysosomes (pH 5, pH 6) and the natural pH (pH 7.4, specific to blood flow). [Figure 3.4] The drug release profiles (17AAG and staurosporine-ST) from PLGA / PVA / LIPO17AAG / ST_2 nanoparticles in PBS buffer pH 7.4 are shown, compared to purified PLGA / PVA nanoparticles loaded with 17AAG and staurosporine (ST) but not subjected to lipid coating. [Figure 3.5] This shows the release profiles of staurosporine (ST) and 17AAG from PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles in PBS7.4 buffer containing 5% BSA at 37°C. [Figure 3.6] This shows the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles after incubation at 37°C for 1 hour with the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.7] This shows the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles after incubation at 37°C for 2 hours with the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.8] This shows the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles after incubation at 37°C for 3 hours with the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.9] This shows the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles after incubation at 37°C for 4 hours with the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.10] This shows the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles after incubation at 37°C for 24 hours with the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.11] This shows the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles after incubation at 37°C for 48 hours with the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.12] This shows the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles after incubation at 37°C for 96 hours with the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.13] This shows the size distribution of the hydrodynamic radius of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles after incubation at 37°C for 120 hours with the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.14] This shows the size distribution of the hydrodynamic radius of PLGA / PVA nanoparticles at T=25°C before the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.15] This shows the size distribution of the hydrodynamic radius of PLGA / PVA nanoparticles at T=25°C after the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.16] This shows the size distribution of the hydrodynamic radius of PLGA / PVA nanoparticles after incubation at T=37°C for 1 hour with the addition of 5% BSA, as measured by dynamic light scattering. [Figure 3.17] This plot shows the ratio of staurosporine encapsulated in PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles to the total drug content in drug solutions stored in PBS buffer pH 7.4, with and without the addition of stabilizing agents. [Figure 3.18]The graph shows the ratio of 17AAG encapsulated in PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles to the total drug content in a drug solution stored in PBS buffer pH 7.4, compared to cases where no stabilizing agent is added. [Figure 3.19] This study demonstrates the dependence of inhibition of proliferation in 8MGBA cells (human brain gliomas) on the concentration of the free drug mixture 17AAG+ staurospoly and the drug encapsulated in PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles. [Figure 3.20] This study demonstrates the dependence of the inhibition of A172 cell proliferation (human glioblastoma multiforme) on the concentration of the free drug mixture 17AAG+ staurospoly and the drug encapsulated in PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles. [Figure 3.21] This study demonstrates the dependence of Hs683 cell (human brain glioma) proliferation inhibition on the concentration of drugs encapsulated in free drug mixtures 17AAG+ staurospoly and PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles. [Figure 3.22] This study demonstrates the dependence of LN229 cell proliferation inhibition in LN229 cells (human brain gliomas) on the concentration of the free drug mixture 17AAG+ staurospoly and the drug encapsulated in PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles. [Figure 3.23] This study demonstrates the dependence of LUDLU-1 cell (human brain glioma) proliferation inhibition on the concentration of the free drug mixture 17AAG+ staurospoly and the drug encapsulated in PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles. [Figure 3.24] This study demonstrates the dependence of NCl-H226 cell proliferation inhibition in human lung squamous cell carcinoma on the concentration of the free drug mixture 17AAG+ staurospoly and the drug encapsulated in PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles. [Figure 3.25] This study demonstrates the dependence of NCl-H520 cell proliferation inhibition in human lung squamous cell carcinoma on the concentration of the free drug mixture 17AAG+ staurospoly and the drug encapsulated in PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles. [Figure 3.26]This study demonstrates the dependence of inhibition of SKMES-1 cell proliferation (human lung squamous cell carcinoma) on the concentration of the free drug mixture 17AAG+ staurospoly and the drug encapsulated in PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles. [Modes for carrying out the invention]

[0025] Table 1 Mass composition and %ww of PLGA / PVA / LIPO support

[0026] [Table 1]

[0027] Table 2 Bulk composition of PLGA / PVA / LIPO nanoparticles and solvents used to form emulsions, as well as the addition of drugs and stabilizers.

[0028] [Table 2-1]

[0029] [Table 2-2]

[0030] [Table 2-3]

[0031] Table 3 Physicochemical parameters of PLGA / PVA / LIPO nanoparticles - size (hydrodynamic radius, HR), polydispersity index (PDI), drug encapsulation rate (%EE), and C / C (c / ml) for nanoparticles with one or more drugs attached. 17AAGST concentration ratio

[0032] [Table 3-1]

[0033] [Table 3-2]

[0034] Example 1: Nanoparticles PLGA / PVA / LIPO / 17AAG / 3G For PLGA / PVA / LIPO / 17AAG / 1G nanoparticles, 200 mg of PVA, 80 mg of PLGA, and 10.0 mg of 17AAG were weighed according to Table 2. The PVA was dissolved in 10 ml of PBS buffer 7.4 using sonication for about 1 hour until the PVA crystals were completely dissolved, and then left in the refrigerator overnight. The PLGA was dissolved in 5 ml of dichloromethane using sonication for 5 seconds. Then the 17AAG was dissolved in the PLGA solution. It was cooled to 4°C (60 minutes in the refrigerator). The dichloromethane solution containing PLGA and 17AAG, followed by the PVA aqueous solution, was then added to a large glass container (50 ml bottle) and cooled again in the refrigerator for 30 minutes. The mixture was then stirred using an ultrasonic probe at 80% amplitude for 1 minute until an emulsion was obtained. The ultrasonic probe was immersed near the interface between the two phases, about 5 mm above the interface. The resulting emulsion was poured into a 100 ml crystallizer along with a stirring element to ensure all of the emulsion was transferred to the crystallizer. The bottle was washed with 2 ml of PBS 7.4 (the washed solution was then placed in the crystallizer). The entire mixture was placed on a magnetic stirrer for 12 hours until the dichloromethane had completely evaporated (400 RPM, 37°C). The solution was diluted to 15 mL with PBS buffer pH 7.4. 1 mL each of PLGA / PVA nanoparticles (unpurified) loaded with 17AAG and 200 μL of liposomes (DPPC / cholesterol / DSPE_PEG(2000)NH2(1 / 0.5 / 0.04)) synthesized in 0.9% NaCl were added to a glass bottle (10 mL) along with a stirring element. The sample was heated in a 45°C water bath for 4 hours, while being stirred with a magnetic stirrer (400 RPM). It was then left in the refrigerator for 12 hours. A 0.5 ml sample was set aside for chromatographic determination of the %EE encapsulation rate using a Sephadex G-25 packed column. The remaining nanoparticles were purified by high-speed centrifugation (15000 RPM, 4 min) and washed twice with PBS7.4 buffer. After each addition of PBS7.4, the nanoparticles were redispersed using sonication for 5 seconds. The zeta potential of the nanoparticles was measured and is shown in Figure 1.1. After the final addition of PBS7.4 buffer, 20 μL of glucose solution (100 mg / ml) was added to each 1 mL of nanoparticles.PLGA / PVA / LIPO / 17AAG / 3G nanoparticles were thus obtained. Next, the size and polydispersity coefficient of the PLGA / PVA / LIPO / 17AAG / 3G nanoparticles were determined using the DLS method. Subsequently, the nanoparticles were sterilized using a sterile PTFE-filled hydrophilic syringe filter with a pore size of 0.45 μm, and their size and PDI were determined again. Figure 1.2 shows the dispersion of the hydrodynamic radius for PLGA / PVA / LIPO / 17AAG / 3G nanoparticles, along with the PDI polydispersity index after the sterilization process.

[0035] The concentration and %EE of the loaded drug were determined by UV-Vis spectroscopy against a calibration curve at a wavelength of 333 nm. Nanoparticle samples were dissolved in DMSO containing hydrochloric acid according to the ratio: 50 μL of sample, 50 μL of 0.3 M HCl, 50 μL of PBS 7.4, and 850 μL of DMSO, and spectra were taken. The spectra are shown in Figures 1.3A and 1.3B.

[0036] In addition, the antiproliferative activity of PLGA / PVA / LIPO / 17AAG / 3G nanoparticles was evaluated in comparison to that of free drugs. In vitro cultured lung cancer and glioma tumor cells were seeded at 1,000 cells per well in sterile 384-well plates. After incubation for 24 hours (37°C, 5% CO2, 95% humidity), cells were treated with at least eight different concentrations of 17AAG solution or PLGA / PVA / LIPO / 17AAG / 3G nanoparticles. Cells were then incubated for 72 hours (37°C, 5% CO2, 95% humidity), and cell proliferation inhibition was evaluated using SRB analysis. Briefly, proteins in each test well were precipitated with 20% trichloroacetic acid solution for 1 hour, washed, and labeled with a 1% acetic acid solution of 0.4% sulforhodamine B (for 30 minutes). After washing off excess dye with a 1% acetic acid solution, the protein-binding dye was dissolved in a 10 mM TRIS buffer solution (for 30 minutes), and the absorbance was read using a spectrophotometer at 540 nm. Growth inhibition was calculated using the following formula:

[0037]

number

[0038] During the ceremony, %Zahlnh - Inhibition of proliferation A m - Absorbance of cell-free wells (culture medium) A k - Absorbance of the control well (cells treated with pure culture medium) A p - Absorbance of wells treated with the compound.

[0039] The results are shown in Figures 1.4 to 1.11. Conclusion: Drug 17-AAG, after being loaded onto artificial nanocarriers, exhibited antiproliferative activity at least comparable to that of the free drug. A significant increase in the activity of compound 17AAG after loading onto nanocarriers was observed for a significant proportion of the lineage, particularly at low drug concentrations, suggesting the possibility of reducing the therapeutic dose of PLGA / PVA / LIPO / 17AAG / 3G nanoparticles compared to free 17AAG while maintaining the required antitumor activity.

[0040] Example 2: PLGA / PVA / LIPO / ST / 4T nanoparticles For PLGA / PVA / LIPO / ST / $G nanoparticles, 200 mg of PVA and 80 mg of PLGA were weighed according to Table 2. The PVA was dissolved in 10 ml of PBS buffer 7.4 using sonication for about 1 hour until the PVA crystals were completely dissolved, and then left in the refrigerator overnight. The PLGA was dissolved in 5 ml of dichloromethane and sonicated for 5 seconds. Next, 10 mg of staurosporine was weighed and dissolved in the PLGA solution. It was cooled to 4°C (30 minutes in the refrigerator). The dichloromethane solution containing PLGA and staurosporine, and the PVA aqueous solution were then added to a large glass container (100 ml bottle) and cooled again in the refrigerator for 30 minutes. The mixture was then stirred using an ultrasonic probe at 80% amplitude for 2 minutes until an emulsion was obtained. The ultrasonic probe was immersed near the interface between the two phases, about 5 mm above the interface. The resulting emulsion was poured into a 50 ml crystallizer along with a stirring element to ensure all of the emulsion was transferred to the crystallizer. The bottle was washed with 2 ml of PBS 7.4 (the washed solution was then placed in the crystallizer). The entire mixture was placed on a magnetic stirrer for 2 hours until the dichloromethane had completely evaporated (400 RPM, 37°C). The solution was diluted to 15 mL with PBS buffer pH 7.4. 1 mL each of staurosporine-loaded PLGA / PVA nanoparticles (unpurified) and then 200 μL of synthesized liposomes (DPPC / cholesterol / DSPE-PEG(2000)NH2(1 / 0.5 / 0.04) 10 mg / ml) in 0.9% NaCl were added to a glass bottle (10 mL) along with a stirring element. The sample was heated in a 60°C water bath for 2 hours, while being stirred with a magnetic stirrer (400 RPM). It was then left in the refrigerator for 14 hours. A 0.5 ml sample was set aside for chromatographic determination of the %EE encapsulation rate using a Sephadex G-25 packed column. The remaining nanoparticles were purified by high-speed centrifugation (15000 RPM, 4 min) and washed twice with PBS7.4 buffer. After each addition of PBS7.4, the nanoparticles were redispersed using sonication for 5 seconds. The zeta potential of the nanoparticles was measured and is shown in Figure 2.1. After the final addition of PBS7.4 buffer, 20 μL of 40% nonionic surfactant solution was added for every 1 mL of nanoparticles.PLGA / PVA / LIPO / ST / 4T nanoparticles were thus obtained. Next, the size and polydispersity coefficient of the PLGA / PVA / LIPO / ST / 4T nanoparticles were determined using the DLS method. Subsequently, the nanoparticles were sterilized using a sterile PTFE-filled hydrophilic syringe filter with a pore size of 0.45 μm, and their size and PDI were determined again. Figure 2.2 shows the dispersion of the hydrodynamic radius for PLGA / PVA / LIPO / ST / 4T nanoparticles, along with the PDI polydispersity index after the sterilization process.

[0041] The concentrations and %EE of the loaded staurosporine were determined using the following method: - UV-Vis spectroscopy for calibration curves at 372, 294, and 353 nm. Nanoparticle samples were dissolved in DMSO containing hydrochloric acid according to the ratio: 25 μL sample, 50 μL 0.3 M HCl, 75 μL PBS7.4, and 850 μL DMSO, and spectra were taken as shown in Figure 2.3.

[0042] - Chromatographic method by purifying a 0.5 ml final PLGA / PVA / LIPO / ST / 4T nanoparticle sample. Purification was performed using a Sephadex G-25 packed chromatography column. After depositing 0.5 ml of unpurified PLGA / PVA / LIPO / ST / 4T nanoparticle sample onto a Sephadex G-25 packed column, eight 1 ml fractions were collected with PBS buffer pH 7.4. The contents of each fraction were examined by UV-Vis spectroscopy. Fractions 3-5 containing PLGA / PVA / LIPO / ST / 4T nanoparticles were combined, and the drug concentration of the nanoparticles was determined by UV-Vis spectroscopy against a calibration curve. PLGA / PVA / LIPO / ST / 4T samples were dissolved in DMSO containing hydrochloric acid according to the ratio of 50 μL of sample, 50 μL of 0.3 M HCl, 50 μL of PBS 7.4, and 850 μL of DMSO. Subsequently, unbound staurosporines were eluted from the chromatography column by washing it with 25 ml of 50% ethanol solution. The concentrations of unbound staurosporines were determined against calibration curves of staurosporines in 50% ethanol at 372, 294, and 353 nm.

[0043] The results of the %EE calculation are summarized in Table 3. In addition, the antiproliferative activity of PLGA / PVA / LIPO / ST / 4T nanoparticles was evaluated in comparison to free staurosporine. In vitro cultured lung cancer and glioma tumor cells were seeded at 1,000 cells per well in sterile 384-well plates. After incubation for 24 hours (37°C, 5% CO2, 95% humidity), cells were treated with at least eight different concentrations of staurosporine solution or PLGA / PVA / LIPO / ST / 4T nanoparticles. Cells were then incubated for 72 hours (37°C, 5% CO2, 95% humidity), and cell proliferation inhibition was evaluated using SRB analysis. Briefly, proteins in each test well were precipitated with 20% trichloroacetic acid solution for 1 hour, washed, and labeled with a 1% acetic acid solution of 0.4% sulforhodamine B (for 30 minutes). After washing off excess dye with a 1% acetic acid solution, the protein-binding dye was dissolved in a 10 mM TRIS buffer solution (for 30 minutes), and the absorbance was read using a spectrophotometer at 540 nm. Growth inhibition was calculated using the following formula:

[0044]

number

[0045] During the ceremony, %Zahlnh - Inhibition of proliferation A m - Absorbance of cell-free wells (culture medium) A k - Absorbance of the control well (cells treated with pure culture medium) A p - Absorbance of wells treated with the compound.

[0046] The results are shown in Figures 2.3 to 2.11. Conclusion: Staurosporines loaded onto artificial nanocarriers exhibited antiproliferative activity at least comparable to that of free staurosporines. In some strains, a significant increase in the activity of compound 17AAG was observed after loading onto nanocarriers, suggesting the possibility of reducing the therapeutic dose of PLGA / PVA / LIPO / ST / 4T compared to free staurosporines while maintaining antitumor activity.

[0047] Example 3: PVA / LIPO / 17AAG / ST polymer nanoparticles loaded with staurosporine and 17AAG and coated with a DPPC / cholesterol / DSPE-PEG(2000)NH2 lipid bilayer. For PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles, 200 mg of PVA, 100 mg of PLGA, 9.5 mg of staurosporine, and 11.5 mg of 17AAG were weighed according to Table 2. The PVA was dissolved in 10 ml of PBS buffer 7.4 using sonication for about 1 hour until the PVA crystals were completely dissolved, and then left in the refrigerator overnight. The PLGA, staurosporine, and 17AAG were dissolved in 5 ml of dichloromethane and sonicated for 5 seconds. This was cooled to 4°C (30 minutes in the refrigerator). Next, the dichloromethane solution containing PLGA, staurosporine, and 17AAG, followed by the PVA aqueous solution, was added to a large glass container (50 ml bottle) and cooled again in the refrigerator for 30 minutes. The mixture was then stirred using an ultrasonic probe at 80% amplitude for 1.5 minutes until an emulsion was obtained. The ultrasonic probe was immersed near the interface between the two phases, approximately 5 mm above the interface. After sonication, a homogeneous emulsion was obtained. The emulsion was poured into a 50 ml crystallizer with a stirring element, ensuring that all of the emulsion was transferred to the crystallizer. The bottle was washed with 2 ml of PBS 7.4 (the washed solution was added to the crystallizer). The entire mixture was placed on a magnetic stirrer for 2 hours (400 RPM, 37°C) until the dichloromethane had completely evaporated. The solution was diluted to 15 mL with PBS buffer pH 7.4. 1 mL each of staurosporine and 17AAG-loaded PLGA / PVA nanoparticles (unpurified) and then 200 μL of synthesized liposomes (DPPC / cholesterol / DSPE-PEG(2000)NH2(1 / 0.5 / 0.04) 10 mg / ml) in 0.9% NaCl were added to four glass bottles (10 mL) with a stirring element. The samples were heated in a 50°C water bath for 3 hours, while being stirred with a magnetic stirrer (400 RPM). They were then left in a refrigerator for 16 hours. 0.5 ml of the sample was set aside for %EE encapsulation determination by chromatography on a Sephadex G-25 packed column. The remaining nanoparticles were purified using high-speed centrifugation (15000 RPM, 4 min) and washed twice with PBS 7.4 buffer. After each addition of PBS 7.4, the nanoparticles were redispersed using sonication for 5 seconds. This yielded PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles.Next, the size and polydispersity coefficient of the PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles were determined using the DLS method. The nanoparticles were then sterilized using a sterile PTFE-filled hydrophilic syringe filter with a pore size of 0.45 μm, and the nanoparticle size, PDI, and zeta potential were determined again. Figure 3.1A shows the distribution of hydrodynamic radii for the PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles, along with the PDI polydispersity index after the sterilization process. Figure 3.1B shows the distribution of zeta potential.

[0048] The concentration and %EE of the administered drug were determined using the following method: - UV-Vis spectroscopy for calibration curves at 372 and 333 nm using the principle of absorbance additivity. Nanoparticle samples were dissolved in DMSO containing hydrochloric acid according to the ratio: 50 μL sample, 50 μL 0.3 M HCl, 50 μL PBS 7.4, and 850 μL DMSO, and spectra were taken.

[0049] - Chromatographic method for purifying a 0.5 ml final PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticle sample. Purification was performed using a Sephadex column while separating the drug. After applying 0.5 ml of unpurified PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticle sample onto a Sephadex G-25 packed column, eight 1 ml fractions were collected in PBS buffer pH 7.4. The contents of each fraction were examined by UV-Vis spectroscopy. Fractions 4-7 containing PLGA / PVA / 17AAG / ST nanoparticles were combined, and the drug concentrations of the nanoparticles were determined by UV-Vis spectroscopy against a calibration curve using the principle of additivity of absorbance. The samples from fractions 4-7 were dissolved in DMSO containing hydrochloric acid according to the ratio of 50 μL of sample, 50 μL of 0.3 M HCl, 50 μL of PBS 7.4, and 850 μL of DMSO. Subsequently, unbound 17AAG was eluted from the chromatography column by washing with 25 ml of PBS buffer pH 7.4, and the concentration of unbound 17AAG was determined at 333 nm relative to the 17AAG calibration curve in PBS buffer. Next, unbound staurosporine was eluted from the column by washing with 25 ml of ethanol:aqueous solution (1:1). The concentration of free staurosporine was determined at 372, 294, and 353 nm relative to the calibration curve by UV-Vis spectroscopy. The results are summarized in Table 3.

[0050] The stability of PLGA / PVA / LIPO / 17AAG / ST nanoparticles was tested over a temperature range of 25–45°C at endosomal / lysosome pH (pH 5, pH 6) and blood flow-specific pH (pH 7.4), after storage at 4°C for 7 days after 24 hours of synthesis and for pH 7.4.

[0051] Figures 3.2 and 3.3 show the dependence of the hydrodynamic diameter (HR) and PDI coefficient of PLGA / PVA / 17AAG / ST / Lipo_2 nanoparticles at a temperature range of 25°C to 45°C in endosomal and lysosome-specific pH buffers (pH 5, pH 6) and innate pH (pH 7.4) 24 hours and 7 days after synthesis (pH 7.4).

[0052] The fact that the hydrodynamic diameter does not change significantly from PDI over the temperature range of 25°C to 45°C at pH 5, 6, and 7.4 demonstrates the stability of PLGA / PVA / 17AAG / ST / Lipo_2 nanoparticles over the tested temperature range, pH, and time.

[0053] Figure 3.4 shows the drug release kinetics of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles in PBS buffer at pH 7.4, compared to purified PLGA / PVA nanoparticles loaded with 17AAG and staurosporine (ST) but not subjected to lipid envelope coating.

[0054] For PLGA / PVA nanoparticles loaded with 17AAG and ST but not subjected to lipid bilayer coating, 17AAG release reached nearly 30% as early as 5 hours and 45% after 24 hours. However, lipid-coated PLGA / PVA / LIPO / 17AAG / ST nanoparticles were characterized by a lack of drug release and sustained release of 17AAG at mean values ​​of 10% after 5 hours, 25% after 24 hours, and 30% after 48 hours. The mean ST release values ​​after 4 hours were independent of the presence of a lipid polymer envelope.

[0055] The effect of proteins on the amount of drug release over time at 37°C was further investigated. Figure 3.5 shows example release profiles of 17AAG and ST in PBS buffer in the presence of 5% BSA, recorded at 37°C.

[0056] In the presence of 5% BSA, PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles showed an average release of 30% of both 17AAG and ST at 24 and 48 hours. At 72 hours, a release of 55% of both drugs was observed. For PLGA / PVA / 17AAG / ST / Lipo_2 nanoparticles, no sudden drug release from the nanoparticles was observed up to 48 hours; the drugs were released gradually.

[0057] To eliminate nanoparticle aggregation during in vitro and in vivo studies, the structural stability of nanoparticles was investigated using PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles as an example in PBS buffer 7.4 supplemented with 5% bovine BSA albumin. Albumin is the most abundant protein in the bloodstream and is used as an additive to cell culture media in in vitro studies. The hydrodynamic radius size distribution of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles at 1, 2, 3, 4, 24, 28, 96, and 120 hours after the addition of 5% bovine albumin is shown in Figures 3.6–3.13. Furthermore, the same test was performed on PLGA / PVA nanoparticles not coated with a lipid bilayer. The hydrodynamic radius distribution of PLGA / PVA liposomes before and after the addition of 10% BSA solution by volume (1:1) is shown in Figures 3.14–3.16, after incubation at 25°C (T=0) and 37°C (1 hour).

[0058] After adding 5% BSA to the PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticle solution, a slight increase in the mean hydrodynamic radius of the nanoparticles was observed, suggesting interaction between the nanoparticles and bovine albumin, as well as the deposition of small amounts of protein on the nanoparticle surface. No large nanostructures were observed, suggesting the stability of the nanoparticles in the presence of plasma proteins. Only after incubation at 37°C for 120 hours in the presence of BSA did signals from a few large structures exceeding 1 μm appear, suggesting an initial loss of stability due to the nanoparticles. The above results suggest that PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles are stable for up to 120 hours in the presence of proteins. In contrast to the lipid-coated nanoparticles, the lipid-uncoated PLGA / PVA nanoparticles aggregated in 5% BSA, suggesting instability in the presence of plasma proteins.

[0059] Stability studies were conducted on PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles during storage in three different solutions: 2 mg / ml glucose solution, 0.8% Tween20 solution, and 0.1 M PBS buffer pH 7.4. Measurements were performed weekly for four consecutive weeks. All collected fractions and test solutions were stored in a refrigerator (4°C to 7°C) for subsequent measurements.

[0060] For each of the three test samples, the drug concentrations (staurosporine and 17-AAG) were determined. For this purpose, a solution containing 50 μl of the test sample, 50 μl of 0.3 M HCl solution, 50 μl of PBS buffer pH 7.4, and 850 μl of DMSO (20-fold dilution) was prepared in an acrylic cuvette. The absorbance of the prepared solution was then recorded using a UV-Vis spectrophotometer. The drug concentrations were determined relative to a calibration curve using the principle of absorbance additiveity.

[0061] In the next step, the hydrodynamic diameter and polydispersity index (PDI) were determined for each of the three test samples using dynamic light scattering (DLS) over a four-week period. The data are summarized in Table 4.

[0062] Table 4. Hydrodynamic diameter and PDI polydispersity coefficient values ​​for PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles measured after storage at 4°C to 7°C for 0, 7, 14, 21, and 28 days with the addition of a stabilizer.

[0063] [Table 4-1]

[0064] [Table 4-2]

[0065] In addition, drug leakage from nanoparticles was examined using chromatography on a Sephadex G-50 bed. A 200 μl sample was applied to a pre-prepared column, and 1 ml was collected in four fractions, 0.5 ml in four fractions, and 1 ml in two fractions, successively into acrylic cuvettes. After collecting these 10 fractions, their absorbance was tested using a UV-Vis spectrophotometer at wavelengths ranging from 220 to 800 nm. Based on the UV-Vis spectrum, the liposome fraction was detected and grouped together. The remaining fraction, containing free drug (17AAG) not bound to nanoparticles, was transferred to a volumetric flask and further extracted from the column with up to 25 ml of PBS buffer pH 7.4. The column was then washed with a 50% ethanol solution to elute the staurosporine-unbound particles from the column. Depending on the sample, 25 ml to 35 ml of solution was collected. The concentration of free staurosporine was then determined against a calibration curve by UV-Vis spectroscopy.

[0066] The drug concentration in the liposome fraction was determined by UV-Vis spectroscopy, similar to the total drug concentration. A solution containing 100 μl of the liposome fraction, 50 μl of 0.3 M HCl solution, and 850 μl of DMSO was prepared in an acrylic cuvette. This solution was mixed using an automated pipette, and the absorbance was then measured. The drug concentration was then determined relative to a calibration curve using the principle of absorbance additiveity.

[0067] The drug content of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles after storage for 0, 7, 14, 21, and 28 days is shown in Figure 3.17 for staurosporine and in Figure 3.18 for 17AAG.

[0068] PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles exhibit a relatively low and acceptable drug leakage rate from the nanoparticles, up to 15% when stored at 4°C to 7°C for up to 4 weeks. Drug leakage from the nanoparticles is eliminated by adding stabilizing substances such as glucose or nonionic surfactants.

[0069] The stability of the physical parameters was confirmed four months after synthesis. The data is summarized in Table 4. Table 4 Physical parameters of PLGA / PVA / LIPO nanoparticles after synthesis and four months after synthesis

[0070] [Table 5]

[0071] In addition, the anti-proliferative activity of PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles was evaluated by comparing it with the free drug mixture 17AAG + staurosporine. In vitro cultured lung cancer and glioma tumor cells were seeded in a sterile 384-well plate at 1,000 cells per well. After incubation for 24 hours (37 °C, 5% CO2, 95% humidity), the cells were treated with at least eight different concentrations of 17AAG + staurosporine drug mixed solution or PLGA / PVA / LIPO / 17AAG / ST_2 nanoparticles and compared with the more potent anti-cancer active drug staurosporine. The cells were then incubated for 72 hours (37 °C, 5% CO2, 95% humidity), and cell growth inhibition was evaluated using SRB analysis. Briefly, the protein in each test well was precipitated with 20% trichloroacetic acid solution for 1 hour, washed, and labeled with a 1% acetic acid solution of 0.4% sulforhodamine B (for 30 minutes). After washing away the excess dye with 1% acetic acid solution, the protein-bound dye was dissolved in 10 mM TRIS buffer solution (for 30 minutes), and the absorbance was read at 540 nm using a spectrophotometer. The following formula was used to calculate the growth inhibition:

[0072] [Equation]

[0073] Where %Zahlnh - Inhibition of growth A m - Absorbance of the well without cells (medium) A k - Absorbance of the control well (cells treated with pure culture medium) A p - Absorbance of the well treated with the compound.

[0074] The results are shown in Figures 3.19 to 3.2. Conclusion: When loaded onto the developed nanocarrier, the drug mixture 17AAG + staurosporine showed significantly higher antiproliferative activity than the free drug mixture 17AAG + staurosporine. This demonstrates the ability to significantly reduce the therapeutic dose of PLGA / PVA / LIPO / 17AAG / ST_2 compared to free staurosporine while maintaining high antitumor activity.

Claims

1. Stable polymer lipid nanoparticles for the binding and delivery of drugs, particularly drugs having anticancer properties, are characterized by comprising a core shell comprising a core containing 16.1 to 30.3% by weight of polylactic acid-glycolic acid copolymer (PLGA) and 60.6 to 75.5% by weight of polyvinyl alcohol (PVA), and a core shell comprising a lipid mixture of 3.66 to 12.6% by weight of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DDPC), 1.85 to 6.29% by weight of cholesterol, and 0.15 to 0.5% by weight of an ammonium salt of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000(DSPE-PEG(2000)NH2)].

2. The nanoparticle according to claim 1, wherein the PLGA is a 50:50 copolymer having a mass Mw of 24,000 to 38,000 g / mol.

3. The nanoparticles according to claim 1, wherein the PVA has a mass Mw of 30,000 to 70,000 g / mol.

4. In the first step, an aqueous PVA solution with a concentration of 15 mg / ml to 20 mg / ml is prepared, followed by a PLGA solution in dichloromethane with a concentration of 10 mg / ml to 20 mg / ml. Then, at least one drug is weighed in an amount necessary to prepare a solution with a concentration in the range of 0.43 mM to 4.30 mM and dissolved in the PLGA solution. After that, both solutions are cooled to 4°C to 7°C, and then 1 part of the PLGA solution in dichloromethane containing the drug is mixed with 2 parts of the aqueous PVA solution in an equal volume ratio of 1:

2. The mixture is then cooled again to 4°C to 7°C, mixed to form an emulsion, the emulsion is poured into a crystallizer along with a stirring element, the residue is washed with PBS buffer pH 7.4 or water in a volume ratio of 1:7.5 to 1:10 of the reaction mixture and the residue is also put into the crystallizer, the dichloromethane is then completely evaporated, and then the core is obtained by replenishing the volume with PBS 7.4 solution or water until a final PLGA concentration of 3.33 mg / ml to 6.66 mg / ml is obtained. In the second step, a core pre-formed with DPPC / cholesterol / DSPE-PEG(2000)NH2 liposomes (1.0 / 0.61 / 0.040) synthesized by the selected method is covered, the formed lipid membrane is hydrated with sterile saline NaCl 0.9% until the total lipid concentration reaches 10 mg / ml, and dispersed by sonication to obtain a DPPC / cholesterol / DSPE-PEG(2000)NH2 liposome solution as an intermediate for further synthesis. Then, the obtained liposomes are added to an unpurified PVA / PLGA polymer core in a volume ratio of liposomes to polymer core of 0.1:1 to 0.3:1, and the whole is then heated at 45 to 60°C for 2 to 4 hours with stirring at 200 to 600 RPM, the obtained PLGA / PVA / LIPO nanoparticles are left at 4°C for 12 to 24 hours, and purified by washing twice with PBS buffer pH 7.

4. A method for producing stable polymer lipid nanoparticles that transport drugs, particularly drugs having anticancer properties.

5. The method according to claim 4, characterized in that the PLGA is a 50:50 copolymer having Mw of 24,000 to 38,000 g / mol.

6. The method according to claim 4, characterized in that the PVA has a mass Mw of 30,000 to 70,000 g / mol.

7. The method according to claim 4, characterized by adding 0.1 M PBS buffer solution pH 7.4 to the PVA solution.

8. The method according to claim 4, characterized in that the internal core polymer is a poly(L-lactic acid-glycolide) copolymer (PLGA).

9. The method according to claim 4, characterized in that the outer polymer of the core is poly(vinyl alcohol) PVA.

10. The method according to claim 4, characterized in that the drug introduced into the core is 5 to 11.5 mg of staurosporine.

11. The method according to claim 4, characterized in that the drug introduced into the core is 5 to 11.5 mg of 17AAG (tanespimycin).

12. The method according to claim 4, wherein staurosporine and 17AAG (tanespimycin) are introduced into the core at a concentration ratio of 17AAG (tanespimycin) to staurosporine of 0.62 to 9.

5.

13. The method according to claim 4, characterized in that the evaporation of dichloromethane is carried out at a temperature of 37°C to 40°C and at a speed of 200 to 600 RPM using a magnetic stirrer.

14. The method according to claim 4, characterized in that the emulsion is obtained by stirring with an ultrasonic probe at an amplitude of 70% to 90% for 1 to 2 minutes.

15. The method according to claim 4, characterized in that the size of nanoparticles is measured using dynamic light scattering to control the quality of the fabricated PLGA / PVA polymer core.

16. The method according to claim 4, characterized in that DPPC / cholesterol / DSPE-PEG(2000)NH2 liposomes are synthesized by hydration after preparing a chloroform solution of a lipid mixture having the composition of 96.0 mM equivalent of DPPC, 58.8 mM equivalent of cholesterol, and 3.84 mM equivalent of DSPE-PEG(2000)NH2.

17. The method according to claim 16, characterized in that, in the hydration method, the solvent is evaporated using a vacuum evaporator or a vacuum dryer.

18. The method according to claim 4, characterized in that DPPC / cholesterol / DSPE-PEG(2000)NH2 liposomes are synthesized by preparing an ethanol solution of a lipid mixture with concentrations of 96.0 mM equivalent of DPPC, 58.8 mM equivalent of cholesterol, and 3.84 mM equivalent of DSPE-PEG(2000)NH2, and then using an ethanol injection method.

19. 。 The method according to claim 18, characterized in that, in the ethanol injection method, an ethanol solution of lipids is added dropwise to sterile physiological saline mixed with a magnetic stirrer to obtain a solution of DPPC / cholesterol / DSPE-PEG(2000)NH2 liposomes having a total lipid concentration of 10 mg / ml as an intermediate for further synthesis.

20. 。 The method according to claim 4, characterized in that the purification of the nanoparticles is performed by high-speed centrifugation at 15,000 RPM for 2 to 6 minutes.

21. The method according to claim 4, wherein after each addition of PBS7.4, the nanoparticles are dispersed using ultrasound for at least 5 seconds.

22. The method according to claim 4, characterized in that, after the final centrifugation of the nanoparticles, a nonionic surfactant is added to form a 40% solution with a volume ratio of 0.02:1 (surfactant:nanoparticles), or glucose is added.

23. The method according to claim 4, characterized in that the drug content, i.e., %EE, is determined by UV-Vis spectrophotometry or LC-MS for the drug alone or a mixture thereof by dissolving PLGA / PVA / LIPO nanoparticles in HCl-containing DMSO according to scheme: 25-75 μL of sample, 50 μL of 0.3 M HCl, 25-75 μL of PBS7.4, and 850 μL of DMSO.

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