WO2022115075A1 - Targeted nanoparticles carrying dual drugs in the treatment of melanoma - Google Patents

Targeted nanoparticles carrying dual drugs in the treatment of melanoma Download PDF

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WO2022115075A1
WO2022115075A1 PCT/TR2021/051146 TR2021051146W WO2022115075A1 WO 2022115075 A1 WO2022115075 A1 WO 2022115075A1 TR 2021051146 W TR2021051146 W TR 2021051146W WO 2022115075 A1 WO2022115075 A1 WO 2022115075A1
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nlc
peg
tyrosine
dspe
doxorubicin
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French (fr)
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Güliz AK DEMİROZ
Şenay ŞANLIER
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Ege Üni̇versi̇tesi̇
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Priority to EP21898836.8A priority Critical patent/EP4247347A4/en
Publication of WO2022115075A1 publication Critical patent/WO2022115075A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to a drug delivery system, lipid nanoparticles containing doxorubicin and trabectedin which are two drugs effective in the treatment of melanoma and tyrosine ligand targeted to LAT-1 for the specific uptake by melanoma cells, developed for use in the health sector in general, and in the pharmaceutical sector in particular.
  • Skin cancer is one of the most common cancers worldwide. Skin malignancy can occur in different forms, including melanoma, squamous cell carcinoma, and basal cell carcinoma. Malignant melanoma is an aggressive type of cancer derived from melanocytes. Cases of melanoma are increasing day by day worldwide. Melanoma accounts for about 4% of all skin cancer cases, with the vast majority (75%) of deaths from skin cancer. New cases of melanoma in the world are reported as 2,8 per 100.000 people and deaths due to melanoma as 0,6 per 100.000 people. 62.000 new cases are detected in Europe each year.
  • the general treatment protocol is operational practice and radiation therapy.
  • chemotherapy drug (dacarbazine, temozolomide, etc.) is preferred as a better alternative in some cases.
  • Patients with melanoma can be treated in case of early diagnosis; however, classical clinical applications cannot be successful in cases diagnosed at a late stage. Survival time varies between 6-10 months or 5 years depending on the location of metastasis in 5-10% of metastatic melanoma cases. It is known that more effective drugs/methods are needed for the treatment of melanoma cancer. Therefore, researchers are developing new methods and drugs based on nanotechnology.
  • nanostructured lipid carriers are prepared and LAT-1 is targeted in previous techniques used in the treatment of melanoma.
  • the combination of doxorubicin and trabectedin and the addition of trabectedin to nanostructured lipid carrier systems were developed within the scope of the invention and will be tried for the first time in the treatment of melanoma.
  • Present invention relates to lipid nanoparticles and method of their preparation which are drug delivery system containing doxorubicin and trabectedin and targeting ligand which are two drugs effective in the treatment of targeted melanoma developed for use in the treatment of melanoma cancer and which meet the aforementioned needs and eliminate all the disadvantages and bring some additional advantages.
  • the primary objective of the invention is to provide localized and effective treatment of melanoma cancer.
  • the drug delivery system structure of the invention is formulated in gel form. Therefore, it allows the localized and effective treatment of melanoma.
  • the side effect from the drugs decreases and the patient's welfare increases in this way.
  • LAT-1 L-aminoacid transporter- 1
  • doxorubicin and trabectedin drugs were synthesized as an innovation in the treatment of melanoma within the scope of the invention.
  • L-type amino acid carrier 1 (LAT-1) carries branched or aromatic amino acids necessary for basic cellular activities such as cellular growth and proliferation. This amino acid carrier can be overexpressed in melanoma cells, in contrast to its limited distribution and low level expression in normal tissues (Higuchi et al, 2019). They were chosen as the target transporter in the invention since melanoma cells overexpress LAT-1.
  • Nanostructured lipid carriers were preferred in the invention in order to increase the low bioavailability of drugs due to their low solubility in water and to contribute to their transdermal passage.
  • DSPE-PEG-Tyrosine conjugate was formed in the invention after the esterification reaction between l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy(polyethylene glycol)-2000] (sodium salt) (DSPE-PEG-COOH) and L-Tyrosine.
  • Nanostructured lipid carriers (NLC) were prepared using sweet almond oil, myristic acid, Tween 80.
  • the DSPE-PEG-Tyrosine conjugate was loaded into the medium during the preparation of the NLC and nanostructured lipid carriers (P-NLC) with DSPE-PEG-Tyrosine conjugate were obtained.
  • Nanostructured lipid carriers (DTP -NLC) carrying DSPE-PEG- Tyrosine loaded with doxorubicin and trabectedin were created by adding doxorubicin (Dox) and trabectedin (Trb) solutions during the preparation of P-NLC.
  • DTP -NLC charged gel was obtained by adding carbopol 940 polymer on DTP -NLC.
  • Nanostructured lipid carriers have been introduced into the gel formulation due to the ease and advantages of topical application. Dermaroller, which contributes to the penetration of drugs through the skin by opening micron holes in the stratum corneum layer of the skin, will be used in vivo applications. Thus, the route of administration for the treatment of melanoma is easy, with few side effects, as well as a targeted drug delivery system with multiple therapeutic effects has been developed with the invention.
  • Figure 1 Process steps in obtaining the nanostructured lipid carrier loaded gel formulation (DTP-NLC gel) carrying DSPE-PEG-Tyrosine loaded with doxorubicin and trabectedin.
  • Figure 2 Synthesis of DSPE-PEG-Tyrosine conjugate by the esterification reaction between DSPE-PEG-COOH and L-Tyrosine.
  • Figure 3 A) FTIR spectrum of DSPE-PEG-Tyrosine, B) E ⁇ -NMR spectrum of DSPE-PEG- Tyrosine, C) C 13 -NMR spectrum of DSPE-PEG-Tyrosine.
  • Figure 4 A) Hydrodynamic size distribution of the NLC structure, B) Zeta potential distribution of the NLC structure.
  • Figure 5 A) FTIR spectrum of NLC structure, B) DSC graph of NLC structure, C) TGA graph of NLC structure, D) SEM image of NLC structure.
  • Figure 6 A) Hydrodynamic size distribution of P-NLC structure, B) Zeta potential distribution of P-NLC structure.
  • Figure 7 A) FTIR spectrum of P-NLC structure, B) DSC graph of P-NLC structure, C) TGA graph of P-NLC structure, D) SEM image of P-NLC structure.
  • Figure 8 A) Hydrodynamic size distribution of the DTP-NLC structure, B) Zeta potential distribution of the DTP-NLC structure.
  • Figure 9 A) FTIR spectrum of DTP-NLC structure, B) DSC graph of DTP-NLC structure, C) TGA graph of DTP-NLC structure, D) SEM image of DTP-NLC structure.
  • Figure 10 A) DSC graph of DTP-NLC gel, B) SEM image of DTP-NLC gel.
  • Figure 11 A) Time-dependent release of free doxorubicin solution in pH 5 and pH 7,4 media B) Time-dependent release of free trabectedin solution in pH 5 and pH 7,4 media.
  • Figure 12 Time-dependent release of doxorubicin from the DTP-NLC sample at pH 5 and pH 7,4 media.
  • Figure 13 Time-dependent release of doxorubicin from DTP-NLC gel sample in pH 5 and pH 7,4 media.
  • the invention is a nanostructured lipid carrier drug delivery system containing doxorubicin and trabectedin, two drugs effective in the treatment of melanoma and a tyrosine ligand targeted to L-aminoacid transporter-1 (LAT-1) for the specific uptake by melanoma cells.
  • LAT-1 L-aminoacid transporter-1
  • Lipid nanoparticles which are drug delivery system containing doxorubicin and trabectedin and targeting ligand, two drugs effective in the treatment of melanoma, were obtained in the invention.
  • Dox and Trb were loaded into nanostructured lipid carriers and turned into a topically applicable gel.
  • the method of preparing the drug delivery system of the invention comprises the process steps; a) Obtaining the DSPE-PEG-Tyrosine conjugate by the esterification reaction between l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (sodium salt) (DSPE-PEG-COOH) and L-Tyrosine, b) Preparing the nanostructured lipid carriers (NLC) with fat, oil, and surfactant, c) Obtaining the nanostructured lipid carriers (P-NLC) with DSPE-PEG-Tyrosine conjugate by loading the DSPE-PEG-Tyrosine conjugate into the medium during the preparation of nanostructured lipid carriers (NLC), d) Creating nanostructured lipid carriers (DTP -NLC) carrying DSPE-PEG-Tyrosine loaded with doxorubicin and trabectedin by adding doxorubicin
  • a tyrosine ligand is inserted into the structure through the COOH group containing DSPE (l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino)-PEG(polyethylene glycol) structure to target the nanocarrier to LAT-1 for specific uptake by melanoma cells.
  • Trabectedin and doxorubicin were loaded into nanostructured lipid transporters known to have high skin penetration and briefly called NLC in the invention.
  • the NLC lipid content provides an advantage during drug loading since the drugs are lipophilic. Drugs with lipophilic character are more encapsulated in the lipid matrix of the NLC structure. NLCs contain drugs with greater efficiency in this way (Burgarelli et al, 2020).
  • NLCs containing dual drugs An excessive amount of LAT-1 expression in the cells was targeted in order for NLCs containing dual drugs to be taken selectively and at a high rate by melanoma cells.
  • the tyrosine at the end of the NLCs acts as a substrate for LAT-1 and facilitates the entry of the nanostructure into the cell thanks to the DSPE-PEG structure. It has been shown in studies that the use of LAT-1 targeted carrier systems in melanoma may be appropriate even though there are not many LAT-1 targeted melanoma studies.
  • NLCs are formulated with carbopolymer gel for topical application, thus allowing targeted and dual drug-containing NLCs to penetrate the skin.
  • LAT-1 targeting was performed by reaction of NLC structure over DSPE-PEG arm using tyrosine ligand in accordance with the object of the invention.
  • the method applied by Jiao et al. was used as the basis for the ester formation reaction (Jiao et al, 2004).
  • d-water distilled water
  • HC1 hydrochloric acid
  • DMAP 4-Dimethylaminopyridine
  • DSPE-PEG-COOH containing 0,005-0,02 mmol PEG on a separate side was dissolved in 0,5-2 mL d-water with the help of an ultrasonic device.
  • 0,005-0,02 mmol DCC was dissolved in 0,5-2 mL d-water:DMSO.
  • Dicyclohexyl carbodiimide (DCC) solution was added on DSPE-PEG-COOH solution and mixed on orbital mixer at 100-600 rpm at room temperature for 30 minutes.
  • the mixture of DSPE-PEG-COOH and DCC solutions was added to the mixture of tyrosine and N,N-dimethyl aminopyridine (DMAP) solutions at the end of the period. The resulting solution was stirred at room temperature for 2 days.
  • DMAP N,N-dimethyl aminopyridine
  • the DSPE-PEG-tyrosine compound obtained was dialyzed against d- water for 24 hours at room temperature. It is aimed to remove tyrosine, DCC, DMAP, which do not participate in the compound structure, by dialysis at this stage.
  • the conjugate was dried in the oven at 20-80°C and stored at +1-8°C for use in subsequent experiments and analyses after dialysis. 13 C NMR analyses were performed with FTIR and 'H NMR in the samples.
  • Nanostructured lipid carriers were synthesized by a combination of various solid and liquid oils and an aqueous phase (Borges et al, 2019; Hajipour et al, 2019; Pinto et al, 2018).
  • Myristic acid 14:0
  • sweet almond oil of vegetable origin was used as liquid oil
  • Tween 80 was used as surfactant.
  • NLC nanostructured lipid carriers
  • DSPE-PEG-Tyrosine conjugate was added to the structures in order to target LAT-1 after determining the optimum synthesis conditions for NLC. 1-4 mg DSPE-PEG-Tyrosine conjugate was added to the aqueous phase and incubated at the same temperature for this purpose.
  • the lipid phase was slowly added into the aqueous phase under the same temperature conditions and it was homogenized by mixing at 8000 rpm for 10 minutes with the help of a high-speed homogenizer. Then, it was dispersed with a sonicator at 70°C for 10 minutes with 10% amplitude. After the structures were cooled to room temperature, they were kept at +4°C for 24 hours for crystallization. Hydrodynamic size, zeta potential load, FTIR, TG, DSC, SEM analyses were performed in P-NLC structures.
  • the lipid phase was slowly added into the aqueous phase under the same temperature conditions and it was homogenized by mixing at 8000 rpm for 10 minutes with the help of a high-speed homogenizer. Then, it was dispersed with a sonicator at 70°C for 10 minutes with 10% amplitude. Nanoparticles loaded with doxorubicin and trabectedin were prepared. After the structures were cooled to room temperature, they were kept at +4°C for 24 hours for crystallization. The nanocarriers were dialyzed for 4 hours at room temperature against 20-80 mL d-water at the end of the period. It was aimed to separate the drugs that were not loaded on the lipid carrier with the dialysis method.
  • Nanostructured lipid carriers (DTP-NLC) carrying DSPE-PEG-Tyrosine loaded with doxorubicin and trabectedin were prepared in this way.
  • Dox and Trb analyses were performed with high performance liquid chromatography technique in the collected dialysis waters. Percentages of drugs encapsulated in nanoparticles were determined based on the amount of drugs not loaded in nanocarriers. Hydrodynamic size, zeta potential load, FTIR, TG, DSC, SEM analyses were performed in DTP-NLC structures.
  • the prepared nanocarrier was mixed with carbopol gel and formulated in accordance with the topical form.
  • Carbopol gel was prepared at a concentration of 1% for this purpose (Ahmed et al, 2019; Malik and Kaur 2018; Yallapu et al, 2015).
  • 50-150 mg carbopol 940 was gradually added to nanostructured lipid carriers carrying DSPE-PEG-Tyrosine loaded with 5- 15 g doxorubicin and trabectedin -and mixed overnight at room temperature at 500-1200 rpm.
  • 70-140 pL TEA was dropped under mechanical stirrer at 900-1500 rpm and the -pH was brought to around 6.
  • Gel containing DTP-NLC (DTP-NLC gel) was prepared. pH, viscosity, hydrodynamic size, zeta potential load, DSC, SEM analyses were performed in DTP-NLC gel.
  • DTP-NLC gel, DTP-NLC and free Dox and Trb mixture were used for drug release study. 10 mM pH 7.4 phosphate buffer and 10 mM pH 5 acetate buffer were prepared as release media. Drug releases were examined by adding DTP-NLC gel, DTP-NLC and Dox and Trb mixture to dialysis membranes. Release media were collected at the 30th minute-55th hours, and fresh buffers at the same temperature were added and synchronous conditions were provided.
  • NLC, P-NLC and DTP-NLC samples were diluted with saline phosphate buffer (PBS) to a concentration of 0,2-0, 8 mg/mL.
  • Fetal bovine serum was diluted 60% (v/v) with PBS.
  • Serum protein and NLC mixtures were prepared so that the ratio of serum proteins to the nanocarrier system was 10:90, 20:80, 40:60, and 60:40 (v/v) and the final volume was 600 pL.
  • Fetal bovine serum and NLC samples were incubated in an orbital mixer at 160 rpm and 37°C for 2 hours (Cole et al., 2011; Semete et al., 2012). 1 hour of centrifugation was performed at 13.000 rpm at the end of the period.
  • the amount of protein in the supernatant samples was determined according to the Bradford method. 2 mL of the prepared Bradford reagent was taken and added to 100 pL of supernatant and the absorbance of the samples were measured at 595 nm wavelength in the UV-Vis spectrophotometer device as a result of incubation at room temperature for 10 minutes. The amount of protein added at the beginning and the amount of protein that is not bound to the nanocarrier were found with the help of the prepared standard graph. The amount of protein bound to the nanocarriers was reached and the binding percentages were calculated by subtracting the amount of unbound protein from the amount of protein added at the beginning.
  • NLC, P-NLC and DTP-NLC samples were diluted with PBS to 1-0,01 mg/mL concentrations.
  • the whole blood taken into the EDTA tube was centrifuged at 3000 rpm for 10 minutes and the erythrocytes were precipitated. Erythrocytes were washed twice with PBS and diluted with PBS to obtain 2% hematocrit.
  • 1 mL of erythrocyte suspension with 1 mL nanocarrier system was incubated in an orbital mixer at 160 rpm and 37°C for 2 hours (Meyer et al., 2016; Yallapu et al., 2015).
  • Erythrocytes were also incubated with PBS as a negative control group at 1:1 (v/v) ratios and with 1% Triton X-100 as a positive control. All samples were centrifuged at 3000 rpm for 10 minutes to precipitate erythrocytes at the end of the incubation. The supernatants were again centrifuged at 13.000 rpm for 1 hour to precipitate the nanocarrier at the end of the centrifugation. Hemoglobin was determined by spectrophotometric measurement of absorbance at 540 nm in the supernatant phase. % hemolysis amounts of erythrocytes were determined over the amount of hemoglobin released as a result of hemolysis.
  • Aromatic proton and amide NH proton signal is observed at 7,94 ppm.
  • the disappearance of phenolic -OH (8,31 ppm) from the tyrosine structure indicates that the DSPE-PEG-Tyrosine synthesis occurs through the water separation by combining the -OH part of the tyrosine structure with the carboxyl group of the DSPE-PEG-COOH molecule.
  • the data obtained were found to be consistent with the literature (Patel et al, 2005).
  • the 13 C-NMR spectrum of DSPE-PEG-Tyrosine is given in Figure 3C.
  • CH 2 and C3 ⁇ 4 carbon signals in the 14,35- 72,72 ppm range seen in the DSPE-PEG-COOH structure are observed (Jeong et al, 1999).
  • the hydrodynamic size distribution of the NLC structure is given in Figure 4A and the zeta potential distribution is given in Figure 4B.
  • the zeta potential value of the NLC structure is - 32,21 ⁇ 1,48 mV and the hydrodynamic size is 62,80 ⁇ 3,55 nm.
  • the DSC graph of the NLC sample is given in Figure 5B.
  • the melting temperature of the NLC sample is 54,99°C. Considering the literature data and explanations, the melting temperature of the NLC sample in the study is appropriate (Badr-Eldin et al, 2019; Gallarate et al, 2009; Jansook et al, 2019; Pinto et al, 2018).
  • the TGA graph of the NLC sample is given in Figure 5C. Weight loss was experienced starting at 120°C up to 250°C and maximum weight loss occurred at 229,91°C in the thermal gravimetric analysis of the NLC sample. In addition, there was another weight loss in the range of 250°C-430°C, where 396,60°C was the peak.
  • Weight loss from 120°C to 250°C is thought to be due to the decomposition of myristic acid and weight loss from 250°C to 430°C is thought to be due to the degradation of sweet almond oil and Tween 80 and results consistent with the literature were obtained (Abbaspourrad 2018; Karimi et al, 2021; Lin et al, 2018; Soleimanifard et al, 2019; Valdes et al, 2015).
  • the SEM image of the NLC sample is given in Figure 5D. It is seen when the SEM image is examined that the NLC dimensions are in the range of 60-70 nm and structures with a morphology close to the globe are obtained.
  • the hydrodynamic size distribution of the P-NLC structure is given in Figure 6A and the zeta potential distribution is given in Figure 6B.
  • the zeta potential value of the P-NLC structure is -30,16 ⁇ 1,45 mV and the hydrodynamic size is 82,03 ⁇ 2,02 nm.
  • the FTIR spectrum of the P-NLC sample is given in Figure 7A. It is seen that the bending signals of CH groups at 1462, 1470 cm-1 and 1375, 1357 cm-1 in the FTIR spectrum of the P- NLC structure increase compared to the NLC sample. It is thought that C-H groups in the DSPE-PEG-Tyrosine structure cause an increase in the P-NLC sample compared to the NLC sample. It can be said that there is an increase in C-OH stress signals at 1166, 1146 and 1090 cm 1 by adding DSPE-PEG-Tyrosine conjugate to the NLC structure in the study. It is thought considering the literature results that DSPE-PEG-Tyrosine participates in the NLC structure and P-NLC synthesis is performed successfully (Haghiral sadat et al, 2018; Marasini et al, 2020).
  • the DSC graph of the P-NLC sample is given in Figure 7B.
  • the melting temperature of the DSPE-PEG-Tyrosine was measured as 47,21°C and the melting temperature of the P-NLC sample as 54,70°C in the DSC graph of the P-NLC structure. It was determined that there was a very small decrease in the melting temperature of the P-NLC structure according to the NLC structure.
  • the TGA graph of the P-NLC sample is given in Figure 7C. It was determined as a result of the TG analysis of the P-NLC sample that the first maximum degradation occurred at 227,20°C and a weight of -22% was degraded at maximum 404,08°C. Some shift in the second step maximum decomposition temperature is thought to be caused by DSPE-PEG- Tyrosine in the structure according to the NLC sample. This result is supported by the fact that the weight loss in this second-line degradation is slightly higher than the NLC sample.
  • the SEM image of the P-NLC sample is given in Figure 7D. It is seen considering the SEM image that the dimensions of the P-NLC are in the range of 75-90 nm and are more regular and global than the NLC samples.
  • the optimum Dox encapsulation percentage in DTP -NLC structures was 60,01 and Trb encapsulation percentage was 95. 3 mg Dox and 95 pg Trb were loaded into the nanocarrier under the specified synthesis conditions.
  • the hydrodynamic size distribution of the DTP -NLC structure is given in Figure 8A and the zeta potential distribution is given in Figure 8B.
  • the zeta potential value of the DTP -NLC structure is -27,89 ⁇ 1,12 mV and the hydrodynamic size is 123,77 ⁇ 9,69 nm.
  • Li et al. showed that negatively charged zeta potential values increased the stability and circulation time of the drug for NLC systems, and also revealed that NLC structures of less than 200 nm accumulated in tumor tissue with the effect of EPR and thus prevented tumor growth.
  • the study also states that dual drug systems are promising (Li et al., 2018). It has been reported by Heurtault et al.
  • the DTP -NLC sample is suitable for use in anticancer treatment in terms of size and zeta potential value.
  • the FTIR spectrum of the DTP -NLC sample is given in Figure 9A. It was determined in the FTIR spectrum of the DTP -NLC structure that the C-H peak intensity, which signals at 3012 cm 1 , increased slightly, although the signals of the lipids generally dominated (overlay). A slight increase was observed in the C-O-C and C-N stretch signals between 1285-1200 cm 1 and the C-0 stretch signal from the primary alcohol groups was exacerbated at 1190 cm 1 . The increase in the FTIR spectrum of these groups in the doxorubicin and trabectedin structures of the DTP-NLC sample indicates that the nanoparticle structures are encapsulated with doxorubicin or doxorubicin and trabectedin. It is thought that this exacerbation may also come from the Trb structure since Trabectedin FTIR analysis cannot be performed.
  • the DSC graph of the DTP-NLC sample is given in Figure 9B.
  • the melting temperature of the DTP-NLC structure was determined as 55,08°C in the DSC graph.
  • a slight increase occurred in the melting temperature of the nanoparticles in the P-NLC sample with the loading of doxorubicin and trabectedin.
  • a very similar graph was obtained to the curve obtained in the doxorubicin and trabectedin-free P-NLC sample when the DSC curve was examined. Therefore, it can be concluded from these results that the drugs are encapsulated in the lipid matrix.
  • the TGA graph of the DTP-NLC sample is given in Figure 9C.
  • a large weight loss was experienced in the first stage in the range of 120°C-245°C and with a maximum temperature of 221°C as a result of the TGA analysis of the DTP-NLC structure.
  • Another degradation occurred in the temperature range of 245°C-450°C, with a peak of 378,53°C, and it was observed that the weight loss in this step increased to -30%.
  • the entire nanoparticle structure is degraded at temperatures after 450°C. It is thought that the decomposition behavior especially in the range of 245°C-450°C changes with the addition of drugs to the structure when compared with the P-NLC sample.
  • the SEM image of the DTP-NLC sample is given in Figure 9D. When looked at the SEM images, it is seen that the dimensions of the DTP-NLC are in the range of 100-120 nm and in the morphology close to the sphere.
  • the pH of DTP-NLC gel is 6,01 ⁇ 0,05. It was determined that the pH value of DTP-NLC charged gel was suitable for dermal use even though there was no difference between the pH values of empty carbopol gel and DTP-NLC charged gel.
  • the viscosity value of DTP-NLC gel is 13689 ⁇ 101,89 cp. There is almost no difference between empty carbopol gel and DTP- NLC charged gel in terms of viscosity, and it can be said that the prepared DTP-NLC gel has an appropriate viscosity that can be applied to the skin surface in the study.
  • the hydrodynamic size of DTP-NLC gel is 149,60 ⁇ 20,93 nm and the zeta potential value is - 34,52 ⁇ 13,16 mV. It is thought that the dimensions may have increased slightly compared to the DTP-NLC particle group with the entry of DTP-NLC particles into the gel structure. It is expected that the zeta potential value will be negative since it is known that carbopol gel is a polyacrylic polymer, and there has been an increase in the absolute value of the negative load with the addition of minus-loaded DTP-NLC to the structure. In addition, there was an increase in the hydrodynamic size of DTP-NLC charged gel compared to DTP-NLC.
  • the DSC graph of the DTP-NLC-loaded gel is given in Figure 10A. It was determined considering the DSC graph that the melting temperature of DTP-NLC charged gel was 116,83°C. It is seen that it reaches a higher melting temperature compared to DTP-NLC and is in accordance with the literature (Khurana et al, 2013; Patil et al, 2015; Wen et al, 2012).
  • the SEM image of the DTP-NLC-loaded gel is given in Figure 10B. Structures with dimensions in the range of 160-200 nm are seen in the morphology close to the sphere when the SEM image of DTP-NLC gel is examined.
  • Time-dependent release of free doxorubicin and trabectedin solutions in pH 5 and pH 7,4 media is shown in Figures 11 A and 1 IB.
  • the time-dependent release of doxorubicin from the DTP-NLC sample at pH 5 and pH 7,4 media is shown in Figure 12.
  • the time-dependent release of doxorubicin from DTP-NLC gel sample in pH 5 and pH 7,4 media is shown in Figure 13. While the total amount of doxorubicin in pH 5 buffer in free form shows release in 1,5 hours, 86,38% at the 1.5 th hour in pH 7,4 buffer and 100% at the 4th hour are observed.
  • the release of trabectedin was 86% at pH 5 and 80% at pH 7,4 at the 4th hour in the free drug solution.
  • Table 1 shows the protein percentages that bind to NLC, P-NLC, DTP-NLC groups as a result of incubation of nanocarriers with fetal bovine serum.

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Abstract

The invention relates to a drug delivery system which is lipid nanoparticles containing doxorubicin and trabectedin, two drugs effective in the treatment of melanoma cancer, and a tyrosine ligand targeted to LAT-1 for the specific uptake by melanoma cells, developed for use in the treatment of melanoma.

Description

TARGETED NANOPARTICLES CARRYING DUAL DRUGS IN THE TREATMENT
OF MELANOMA
Technical Field Related to the Invention
The invention relates to a drug delivery system, lipid nanoparticles containing doxorubicin and trabectedin which are two drugs effective in the treatment of melanoma and tyrosine ligand targeted to LAT-1 for the specific uptake by melanoma cells, developed for use in the health sector in general, and in the pharmaceutical sector in particular.
State of the Art of the Invention (Prior Art)
Skin cancer is one of the most common cancers worldwide. Skin malignancy can occur in different forms, including melanoma, squamous cell carcinoma, and basal cell carcinoma. Malignant melanoma is an aggressive type of cancer derived from melanocytes. Cases of melanoma are increasing day by day worldwide. Melanoma accounts for about 4% of all skin cancer cases, with the vast majority (75%) of deaths from skin cancer. New cases of melanoma in the world are reported as 2,8 per 100.000 people and deaths due to melanoma as 0,6 per 100.000 people. 62.000 new cases are detected in Europe each year.
The general treatment protocol is operational practice and radiation therapy. In addition, chemotherapy (dacarbazine, temozolomide, etc.) is preferred as a better alternative in some cases. Patients with melanoma can be treated in case of early diagnosis; however, classical clinical applications cannot be successful in cases diagnosed at a late stage. Survival time varies between 6-10 months or 5 years depending on the location of metastasis in 5-10% of metastatic melanoma cases. It is known that more effective drugs/methods are needed for the treatment of melanoma cancer. Therefore, researchers are developing new methods and drugs based on nanotechnology.
There are studies including combinations of different chemotherapy drugs such as vemurafenib and carboplatin used in the treatment of melanoma in the state of the art. Trabectedin is known to have a synergistic effect when used with doxorubicin. In addition, the in vitro and in vivo effectiveness of both drugs in the treatment of melanoma has been demonstrated in studies. It is known that topical administration of chemotherapy drugs in the treatment of melanoma will also provide an advantage.
In addition, studies conducted with doxorubicin and trabectedin have been found to be associated with ovarian cancer and soft tissue sarcomas.
There is no method in the literature in which nanostructured lipid carriers are prepared and LAT-1 is targeted in previous techniques used in the treatment of melanoma. The combination of doxorubicin and trabectedin and the addition of trabectedin to nanostructured lipid carrier systems were developed within the scope of the invention and will be tried for the first time in the treatment of melanoma. It is a unique drug delivery system that can be used in the treatment of melanoma of nanostructured lipid transporters containing LAT-1 -targeted doxorubicin and trabectedin developed by the invention since the levels of LAT-1 increased in melanoma, the effectiveness of doxorubicin and trabectedin in melanoma is known to be effective for topical administration of nanostructured lipid transporters and gel formulation.
Briefly, there is no drug delivery system containing lipid nanoparticles containing doxorubicin, trabectedin and targeting ligand, two drugs effective in the treatment of targeted melanoma cancer developed for use in the treatment of melanoma cancer in the prior art.
Brief Description and Objectives of the Invention
Present invention relates to lipid nanoparticles and method of their preparation which are drug delivery system containing doxorubicin and trabectedin and targeting ligand which are two drugs effective in the treatment of targeted melanoma developed for use in the treatment of melanoma cancer and which meet the aforementioned needs and eliminate all the disadvantages and bring some additional advantages.
The primary objective of the invention is to provide localized and effective treatment of melanoma cancer. The drug delivery system structure of the invention is formulated in gel form. Therefore, it allows the localized and effective treatment of melanoma. The side effect from the drugs decreases and the patient's welfare increases in this way.
Nanostructured lipid carriers with LAT-1 (L-aminoacid transporter- 1) target and containing doxorubicin and trabectedin drugs were synthesized as an innovation in the treatment of melanoma within the scope of the invention. L-type amino acid carrier 1 (LAT-1) carries branched or aromatic amino acids necessary for basic cellular activities such as cellular growth and proliferation. This amino acid carrier can be overexpressed in melanoma cells, in contrast to its limited distribution and low level expression in normal tissues (Higuchi et al, 2019). They were chosen as the target transporter in the invention since melanoma cells overexpress LAT-1.
Nanostructured lipid carriers were preferred in the invention in order to increase the low bioavailability of drugs due to their low solubility in water and to contribute to their transdermal passage. DSPE-PEG-Tyrosine conjugate was formed in the invention after the esterification reaction between l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy(polyethylene glycol)-2000] (sodium salt) (DSPE-PEG-COOH) and L-Tyrosine. Nanostructured lipid carriers (NLC) were prepared using sweet almond oil, myristic acid, Tween 80. The DSPE-PEG-Tyrosine conjugate was loaded into the medium during the preparation of the NLC and nanostructured lipid carriers (P-NLC) with DSPE-PEG-Tyrosine conjugate were obtained. Nanostructured lipid carriers (DTP -NLC) carrying DSPE-PEG- Tyrosine loaded with doxorubicin and trabectedin were created by adding doxorubicin (Dox) and trabectedin (Trb) solutions during the preparation of P-NLC. DTP -NLC charged gel was obtained by adding carbopol 940 polymer on DTP -NLC. A flowchart of the described method steps is depicted in Figure 1.
Nanostructured lipid carriers have been introduced into the gel formulation due to the ease and advantages of topical application. Dermaroller, which contributes to the penetration of drugs through the skin by opening micron holes in the stratum corneum layer of the skin, will be used in vivo applications. Thus, the route of administration for the treatment of melanoma is easy, with few side effects, as well as a targeted drug delivery system with multiple therapeutic effects has been developed with the invention.
Definitions of Figures Describing the Invention
The figures and related descriptions required to better understand the invention are as follows.
Figure 1: Process steps in obtaining the nanostructured lipid carrier loaded gel formulation (DTP-NLC gel) carrying DSPE-PEG-Tyrosine loaded with doxorubicin and trabectedin.
Figure 2: Synthesis of DSPE-PEG-Tyrosine conjugate by the esterification reaction between DSPE-PEG-COOH and L-Tyrosine.
Figure 3: A) FTIR spectrum of DSPE-PEG-Tyrosine, B) E^-NMR spectrum of DSPE-PEG- Tyrosine, C) C13-NMR spectrum of DSPE-PEG-Tyrosine.
Figure 4: A) Hydrodynamic size distribution of the NLC structure, B) Zeta potential distribution of the NLC structure.
Figure 5: A) FTIR spectrum of NLC structure, B) DSC graph of NLC structure, C) TGA graph of NLC structure, D) SEM image of NLC structure.
Figure 6: A) Hydrodynamic size distribution of P-NLC structure, B) Zeta potential distribution of P-NLC structure.
Figure 7: A) FTIR spectrum of P-NLC structure, B) DSC graph of P-NLC structure, C) TGA graph of P-NLC structure, D) SEM image of P-NLC structure.
Figure 8: A) Hydrodynamic size distribution of the DTP-NLC structure, B) Zeta potential distribution of the DTP-NLC structure.
Figure 9: A) FTIR spectrum of DTP-NLC structure, B) DSC graph of DTP-NLC structure, C) TGA graph of DTP-NLC structure, D) SEM image of DTP-NLC structure. Figure 10: A) DSC graph of DTP-NLC gel, B) SEM image of DTP-NLC gel.
Figure 11: A) Time-dependent release of free doxorubicin solution in pH 5 and pH 7,4 media B) Time-dependent release of free trabectedin solution in pH 5 and pH 7,4 media.
Figure 12: Time-dependent release of doxorubicin from the DTP-NLC sample at pH 5 and pH 7,4 media.
Figure 13: Time-dependent release of doxorubicin from DTP-NLC gel sample in pH 5 and pH 7,4 media.
Detailed Description of the Invention
The invention is a nanostructured lipid carrier drug delivery system containing doxorubicin and trabectedin, two drugs effective in the treatment of melanoma and a tyrosine ligand targeted to L-aminoacid transporter-1 (LAT-1) for the specific uptake by melanoma cells.
The drug delivery system and its preparation method are described only for clarifying the subject matter in a manner such that no limiting effect is created in this detailed description.
Lipid nanoparticles, which are drug delivery system containing doxorubicin and trabectedin and targeting ligand, two drugs effective in the treatment of melanoma, were obtained in the invention. Dox and Trb were loaded into nanostructured lipid carriers and turned into a topically applicable gel.
The method of preparing the drug delivery system of the invention comprises the process steps; a) Obtaining the DSPE-PEG-Tyrosine conjugate by the esterification reaction between l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (sodium salt) (DSPE-PEG-COOH) and L-Tyrosine, b) Preparing the nanostructured lipid carriers (NLC) with fat, oil, and surfactant, c) Obtaining the nanostructured lipid carriers (P-NLC) with DSPE-PEG-Tyrosine conjugate by loading the DSPE-PEG-Tyrosine conjugate into the medium during the preparation of nanostructured lipid carriers (NLC), d) Creating nanostructured lipid carriers (DTP -NLC) carrying DSPE-PEG-Tyrosine loaded with doxorubicin and trabectedin by adding doxorubicin (Dox) and trabectedin (Trb) solutions during the preparation of P-NLC.
A tyrosine ligand is inserted into the structure through the COOH group containing DSPE (l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino)-PEG(polyethylene glycol) structure to target the nanocarrier to LAT-1 for specific uptake by melanoma cells. Trabectedin and doxorubicin were loaded into nanostructured lipid transporters known to have high skin penetration and briefly called NLC in the invention. The NLC lipid content provides an advantage during drug loading since the drugs are lipophilic. Drugs with lipophilic character are more encapsulated in the lipid matrix of the NLC structure. NLCs contain drugs with greater efficiency in this way (Burgarelli et al, 2020). An excessive amount of LAT-1 expression in the cells was targeted in order for NLCs containing dual drugs to be taken selectively and at a high rate by melanoma cells. The tyrosine at the end of the NLCs acts as a substrate for LAT-1 and facilitates the entry of the nanostructure into the cell thanks to the DSPE-PEG structure. It has been shown in studies that the use of LAT-1 targeted carrier systems in melanoma may be appropriate even though there are not many LAT-1 targeted melanoma studies. NLCs are formulated with carbopolymer gel for topical application, thus allowing targeted and dual drug-containing NLCs to penetrate the skin. It is aimed to be easily topically applied by the patient and/or specialist health professional in the treatment of melanoma with the preparation of the gel formulation of NLCs. It will also be ensured in the invention that the penetration of the NLC gel formulation through the skin will increase by using dermaroller. Dermaroller can also be used very easily by the person through driving.
1. Preparation of LAT-1 targeted nanostructured lipid carriers and loading of trabectedin with doxorubicin 1.1 Synthesis of DSPE-PEG-Tyrosine compound
LAT-1 targeting was performed by reaction of NLC structure over DSPE-PEG arm using tyrosine ligand in accordance with the object of the invention. The method applied by Jiao et al. was used as the basis for the ester formation reaction (Jiao et al, 2004). First of all, 0,01- 0,1 mmol L-tyrosine was dissolved in 10-15 mL distilled water (d-water) from 100-300 pL 1- 5 M hydrochloric acid (HC1) solution and under heating conditions in a magnetic stirrer. 0,5-2 mL of d-water dissolved 0,01-0,04 mmol 4-Dimethylaminopyridine (DMAP) was added and mixed at room temperature for 30 minutes. DSPE-PEG-COOH containing 0,005-0,02 mmol PEG on a separate side was dissolved in 0,5-2 mL d-water with the help of an ultrasonic device. 0,005-0,02 mmol DCC was dissolved in 0,5-2 mL d-water:DMSO. Dicyclohexyl carbodiimide (DCC) solution was added on DSPE-PEG-COOH solution and mixed on orbital mixer at 100-600 rpm at room temperature for 30 minutes. The mixture of DSPE-PEG-COOH and DCC solutions was added to the mixture of tyrosine and N,N-dimethyl aminopyridine (DMAP) solutions at the end of the period. The resulting solution was stirred at room temperature for 2 days. The DSPE-PEG-tyrosine compound obtained was dialyzed against d- water for 24 hours at room temperature. It is aimed to remove tyrosine, DCC, DMAP, which do not participate in the compound structure, by dialysis at this stage. The conjugate was dried in the oven at 20-80°C and stored at +1-8°C for use in subsequent experiments and analyses after dialysis. 13C NMR analyses were performed with FTIR and 'H NMR in the samples.
1.2 Preparation and characterization of nanostructured lipid carriers
Nanostructured lipid carriers were synthesized by a combination of various solid and liquid oils and an aqueous phase (Borges et al, 2019; Hajipour et al, 2019; Pinto et al, 2018). Myristic acid (14:0) was used as fat and sweet almond oil of vegetable origin was used as liquid oil, and Tween 80 was used as surfactant. First, nanostructured lipid carriers (NLC) were started to be prepared without adding the DSPE-PEG-Tyrosine compound to the medium and the synthesis conditions were optimized. Synthesis was performed using lipid phase 4%, Tween 80% 2,5% and water 93,5% by weight (Pinto et al, 2018). 20-60 mg of myristic acid and 50-100 mg of sweet almond oil were weighed into a tube and melted in a water bath at a temperature higher than the melting temperatures, at 70°C. In addition, an aqueous phase solution was prepared using 50-100 mg tween 80 and 2-5 g d-water. The lipid phase was slowly added into the aqueous phase under the same temperature conditions and it was homogenized by mixing at 8000 rpm for 10 minutes with the help of a high-speed homogenizer. Then, it was dispersed with a sonicator at 70°C for 10 minutes with 10% amplitude. After the structures were cooled to room temperature, they were kept at +4°C for 24 hours for crystallization (Luo et al, 2011). Hydrodynamic size, zeta potential load, FTIR, thermal gravimetric (TG), differential scanning calorimeter (DSC), scanning electron microscopy (SEM) analyzes were performed in nanoparticles.
1.3 Preparation and characterization of LAT-1 targeted nanostructured lipid carriers
20-60 mg of myristic acid and 50-100 mg of sweet almond oil were weighed into a tube and melted in a water bath at a temperature higher than the melting temperatures, at 70°C. In addition, an aqueous solution was prepared with 50-100 mg tween 80 and 2-5 g d-water. DSPE-PEG-Tyrosine conjugate was added to the structures in order to target LAT-1 after determining the optimum synthesis conditions for NLC. 1-4 mg DSPE-PEG-Tyrosine conjugate was added to the aqueous phase and incubated at the same temperature for this purpose. The lipid phase was slowly added into the aqueous phase under the same temperature conditions and it was homogenized by mixing at 8000 rpm for 10 minutes with the help of a high-speed homogenizer. Then, it was dispersed with a sonicator at 70°C for 10 minutes with 10% amplitude. After the structures were cooled to room temperature, they were kept at +4°C for 24 hours for crystallization. Hydrodynamic size, zeta potential load, FTIR, TG, DSC, SEM analyses were performed in P-NLC structures.
1.4 Preparation and characterization of LAT-1 targeted doxorubicin and trabectedin loaded nanostructured lipid carriers
20-60 mg of myristic acid and 50-100 mg of sweet almond oil were weighed into a tube and melted in a water bath at a temperature higher than the melting temperatures, at 70°C. 1-10 mg/mL doxorubicin solution dissolved in 80-130 pL DMSO and 40-180 pg/mL trabectedin solution prepared in 80-130 pL DMSO were added to the lipid phase and dissolved in a water bath at 70°C for the encapsulation of the drugs. In addition, an aqueous solution was prepared with 50-100 mg tween 80, 1-4 mg DSPE-PEG-Tyrosine conjugate and 2-5 g d-water. The lipid phase was slowly added into the aqueous phase under the same temperature conditions and it was homogenized by mixing at 8000 rpm for 10 minutes with the help of a high-speed homogenizer. Then, it was dispersed with a sonicator at 70°C for 10 minutes with 10% amplitude. Nanoparticles loaded with doxorubicin and trabectedin were prepared. After the structures were cooled to room temperature, they were kept at +4°C for 24 hours for crystallization. The nanocarriers were dialyzed for 4 hours at room temperature against 20-80 mL d-water at the end of the period. It was aimed to separate the drugs that were not loaded on the lipid carrier with the dialysis method. Nanostructured lipid carriers (DTP-NLC) carrying DSPE-PEG-Tyrosine loaded with doxorubicin and trabectedin were prepared in this way. Dox and Trb analyses were performed with high performance liquid chromatography technique in the collected dialysis waters. Percentages of drugs encapsulated in nanoparticles were determined based on the amount of drugs not loaded in nanocarriers. Hydrodynamic size, zeta potential load, FTIR, TG, DSC, SEM analyses were performed in DTP-NLC structures.
2. Formulation, characterization, and drug release studies of LAT-1 targeted and doxorubicin and trabectedin loaded nanostructured lipid carriers in gel structure
2.1 Gel formulation and characterization
The prepared nanocarrier was mixed with carbopol gel and formulated in accordance with the topical form. Carbopol gel was prepared at a concentration of 1% for this purpose (Ahmed et al, 2019; Malik and Kaur 2018; Yallapu et al, 2015). Firstly, 50-150 mg carbopol 940 was gradually added to nanostructured lipid carriers carrying DSPE-PEG-Tyrosine loaded with 5- 15 g doxorubicin and trabectedin -and mixed overnight at room temperature at 500-1200 rpm. Then, 70-140 pL TEA was dropped under mechanical stirrer at 900-1500 rpm and the -pH was brought to around 6. Gel containing DTP-NLC (DTP-NLC gel) was prepared. pH, viscosity, hydrodynamic size, zeta potential load, DSC, SEM analyses were performed in DTP-NLC gel.
2.2 Drug release study DTP-NLC gel, DTP-NLC and free Dox and Trb mixture were used for drug release study. 10 mM pH 7.4 phosphate buffer and 10 mM pH 5 acetate buffer were prepared as release media. Drug releases were examined by adding DTP-NLC gel, DTP-NLC and Dox and Trb mixture to dialysis membranes. Release media were collected at the 30th minute-55th hours, and fresh buffers at the same temperature were added and synchronous conditions were provided.
3. Biocompatibility Studies
3.1 Protein Binding
NLC, P-NLC and DTP-NLC samples were diluted with saline phosphate buffer (PBS) to a concentration of 0,2-0, 8 mg/mL. Fetal bovine serum was diluted 60% (v/v) with PBS. Serum protein and NLC mixtures were prepared so that the ratio of serum proteins to the nanocarrier system was 10:90, 20:80, 40:60, and 60:40 (v/v) and the final volume was 600 pL. Fetal bovine serum and NLC samples were incubated in an orbital mixer at 160 rpm and 37°C for 2 hours (Cole et al., 2011; Semete et al., 2012). 1 hour of centrifugation was performed at 13.000 rpm at the end of the period. The amount of protein in the supernatant samples was determined according to the Bradford method. 2 mL of the prepared Bradford reagent was taken and added to 100 pL of supernatant and the absorbance of the samples were measured at 595 nm wavelength in the UV-Vis spectrophotometer device as a result of incubation at room temperature for 10 minutes. The amount of protein added at the beginning and the amount of protein that is not bound to the nanocarrier were found with the help of the prepared standard graph. The amount of protein bound to the nanocarriers was reached and the binding percentages were calculated by subtracting the amount of unbound protein from the amount of protein added at the beginning.
3.2 Hemolysis
NLC, P-NLC and DTP-NLC samples were diluted with PBS to 1-0,01 mg/mL concentrations. On the other hand, the whole blood taken into the EDTA tube was centrifuged at 3000 rpm for 10 minutes and the erythrocytes were precipitated. Erythrocytes were washed twice with PBS and diluted with PBS to obtain 2% hematocrit. 1 mL of erythrocyte suspension with 1 mL nanocarrier system was incubated in an orbital mixer at 160 rpm and 37°C for 2 hours (Meyer et al., 2016; Yallapu et al., 2015). Erythrocytes were also incubated with PBS as a negative control group at 1:1 (v/v) ratios and with 1% Triton X-100 as a positive control. All samples were centrifuged at 3000 rpm for 10 minutes to precipitate erythrocytes at the end of the incubation. The supernatants were again centrifuged at 13.000 rpm for 1 hour to precipitate the nanocarrier at the end of the centrifugation. Hemoglobin was determined by spectrophotometric measurement of absorbance at 540 nm in the supernatant phase. % hemolysis amounts of erythrocytes were determined over the amount of hemoglobin released as a result of hemolysis.
Results and Interpretation
1. Preparation of LAT-1 targeted nanostructured lipid carriers and loading of trabectedin with doxorubicin
1.1 Synthesis of DSPE-PEG-Tyrosine compound
Synthesis of DSPE-PEG-Tyrosine conjugate by the esterification reaction between DSPE- PEG-COOH and L-Tyrosine is schematized in Figure 2. The FTIR spectrum of DSPE-PEG- Tyrosine is shown in Figure 3 A. It is thought when the spectrum of DSPE-PEG-Tyrosine is examined that the NH stress from the DSPE-PEG-COOH structure shifts to 3320 cm 1 with the reaction of the structure with the tyrosine. DSPE-PEG-Tyrosine structure shows -CH stresses from DSPE-PEG-COOH structure in the range of 3000-2800 cm 1. The peak observed at 1713 cm 1 in the DSPE-PEG-COOH structure is lost in the DSPE-PEG-Tyrosine structure, so it can be clearly said that the carboxyl end of the DSPE-PEG-COOH structure reacts. The ester bond carbonyl stretch determined at 1737 cm 1 is observed in the DSPE-PEG-Tyrosine structure. An aromatic ring C=C bond stress signal was detected at 1597 cm 1 unlike DSPE- PEG-COOH structure in DSPE-PEG-Tyrosine structure. An aromatic ring C=C bond tension was found as flattened at 1511 cm 1. The formation of these signals also confirms that tyrosine is added to the structure. COO symmetric stress at 1412 cm 1 in the tyrosine structure was determined as DSPE-PEG-Tyrosine increased signal. All these bands also confirm that tyrosine is added to the DSPE-PEG-COOH structure. 'H-NMR spectrum of DSPE-PEG-Tyrosine is given in Figure 3B. When the 'H-NMR spectrum of DSPE-PEG-Tyrosine was examined, an aliphatic CEB, CEE and CH proton resonance signal was detected at 0,85-0,82; 1,22; 3,49; 3,68-3,63, and 4,12 ppm as in DSPE- PEG-COOH and tyrosine structures. Aromatic proton and amide NH proton signal is observed at 7,94 ppm. In addition, the disappearance of phenolic -OH (8,31 ppm) from the tyrosine structure indicates that the DSPE-PEG-Tyrosine synthesis occurs through the water separation by combining the -OH part of the tyrosine structure with the carboxyl group of the DSPE-PEG-COOH molecule. The data obtained were found to be consistent with the literature (Patel et al, 2005).
The 13C-NMR spectrum of DSPE-PEG-Tyrosine is given in Figure 3C. When the DSPE- PEG-Tyrosine 13C-NMR spectrum is examined, CH2 and C¾ carbon signals in the 14,35- 72,72 ppm range seen in the DSPE-PEG-COOH structure are observed (Jeong et al, 1999). There are carbon signals belonging to esteric, carboxyl and ketone groups in the range of 169,71 and 172,98 ppm (Paquin et al, 2015). In the Tyrosine 13C-NMR spectrum, aromatic carbon peaks were found in the range of 115,83-156,90 ppm in accordance with the literature (Anandan et al, 2012; Kulikov et al, 2017). All these peaks are also characteristic in the DSPE-PEG-Tyrosine spectrum. The presence of 5 carbon signals of esteric, carboxyl and ketone carbons in the range of 196,83-169,68 ppm, 4 carbon signals of tyrosine ring in the range of 159,55-116,46 ppm and the presence of signals of (-CH2 + -CH3) carbons in the range of 72,75-14,38 ppm confirmed the DSPE-PEG-Tyrosine structure.
1.2 Preparation and characterization of nanostructured lipid carriers
The hydrodynamic size distribution of the NLC structure is given in Figure 4A and the zeta potential distribution is given in Figure 4B. The zeta potential value of the NLC structure is - 32,21 ±1,48 mV and the hydrodynamic size is 62,80±3,55 nm.
The FTIR spectrum of the NLC structure is given in Figure 5A. It is seen when the FTIR spectrum is examined that there are stress bands belonging to C-H groups of myristic acid, which is the NLC component, at 3100-2800 cm 1. It is thought that the ester bond C=0 stretch signal determined at 1743 cm 1 may come from almond oil and Tween-80 structure as NLC components. It can be said that the sharp peak at 1699 cm 1 belongs to the carboxyl group C=0 stress in the myristic acid structure and the small band at 1161 cm 1 is the C-OH stress band due to the almond oil content. It is seen when all these results are examined that there are IR signals originating from the components, mainly myristic acid, in the NLC structure (Beltran et al, 2011; Brandelero et al, 2012; Ribeiro et al, 2017; Saupe et al, 2006).
The DSC graph of the NLC sample is given in Figure 5B. The melting temperature of the NLC sample is 54,99°C. Considering the literature data and explanations, the melting temperature of the NLC sample in the study is appropriate (Badr-Eldin et al, 2019; Gallarate et al, 2009; Jansook et al, 2019; Pinto et al, 2018).
The TGA graph of the NLC sample is given in Figure 5C. Weight loss was experienced starting at 120°C up to 250°C and maximum weight loss occurred at 229,91°C in the thermal gravimetric analysis of the NLC sample. In addition, there was another weight loss in the range of 250°C-430°C, where 396,60°C was the peak. Weight loss from 120°C to 250°C is thought to be due to the decomposition of myristic acid and weight loss from 250°C to 430°C is thought to be due to the degradation of sweet almond oil and Tween 80 and results consistent with the literature were obtained (Abbaspourrad 2018; Karimi et al, 2021; Lin et al, 2018; Soleimanifard et al, 2019; Valdes et al, 2015).
The SEM image of the NLC sample is given in Figure 5D. It is seen when the SEM image is examined that the NLC dimensions are in the range of 60-70 nm and structures with a morphology close to the globe are obtained.
1.3 Preparation and characterization of LAT-1 targeted nanostructured lipid carriers
The hydrodynamic size distribution of the P-NLC structure is given in Figure 6A and the zeta potential distribution is given in Figure 6B. The zeta potential value of the P-NLC structure is -30,16±1,45 mV and the hydrodynamic size is 82,03±2,02 nm.
The FTIR spectrum of the P-NLC sample is given in Figure 7A. It is seen that the bending signals of CH groups at 1462, 1470 cm-1 and 1375, 1357 cm-1 in the FTIR spectrum of the P- NLC structure increase compared to the NLC sample. It is thought that C-H groups in the DSPE-PEG-Tyrosine structure cause an increase in the P-NLC sample compared to the NLC sample. It can be said that there is an increase in C-OH stress signals at 1166, 1146 and 1090 cm 1 by adding DSPE-PEG-Tyrosine conjugate to the NLC structure in the study. It is thought considering the literature results that DSPE-PEG-Tyrosine participates in the NLC structure and P-NLC synthesis is performed successfully (Haghiral sadat et al, 2018; Marasini et al, 2020).
The DSC graph of the P-NLC sample is given in Figure 7B. The melting temperature of the DSPE-PEG-Tyrosine was measured as 47,21°C and the melting temperature of the P-NLC sample as 54,70°C in the DSC graph of the P-NLC structure. It was determined that there was a very small decrease in the melting temperature of the P-NLC structure according to the NLC structure.
The TGA graph of the P-NLC sample is given in Figure 7C. It was determined as a result of the TG analysis of the P-NLC sample that the first maximum degradation occurred at 227,20°C and a weight of -22% was degraded at maximum 404,08°C. Some shift in the second step maximum decomposition temperature is thought to be caused by DSPE-PEG- Tyrosine in the structure according to the NLC sample. This result is supported by the fact that the weight loss in this second-line degradation is slightly higher than the NLC sample.
The SEM image of the P-NLC sample is given in Figure 7D. It is seen considering the SEM image that the dimensions of the P-NLC are in the range of 75-90 nm and are more regular and global than the NLC samples.
1.4 Preparation and characterization of LAT-1 targeted doxorubicin and trabectedin loaded nanostructured lipid carriers
The optimum Dox encapsulation percentage in DTP -NLC structures was 60,01 and Trb encapsulation percentage was 95. 3 mg Dox and 95 pg Trb were loaded into the nanocarrier under the specified synthesis conditions.
The hydrodynamic size distribution of the DTP -NLC structure is given in Figure 8A and the zeta potential distribution is given in Figure 8B. The zeta potential value of the DTP -NLC structure is -27,89±1,12 mV and the hydrodynamic size is 123,77±9,69 nm. Li et al. showed that negatively charged zeta potential values increased the stability and circulation time of the drug for NLC systems, and also revealed that NLC structures of less than 200 nm accumulated in tumor tissue with the effect of EPR and thus prevented tumor growth. The study also states that dual drug systems are promising (Li et al., 2018). It has been reported by Heurtault et al. that nanoparticles with high zeta potential remain more stable (Heurtault et al., 2003). Therefore, it can be said that the DTP -NLC sample is suitable for use in anticancer treatment in terms of size and zeta potential value.
The FTIR spectrum of the DTP -NLC sample is given in Figure 9A. It was determined in the FTIR spectrum of the DTP -NLC structure that the C-H peak intensity, which signals at 3012 cm 1, increased slightly, although the signals of the lipids generally dominated (overlay). A slight increase was observed in the C-O-C and C-N stretch signals between 1285-1200 cm 1 and the C-0 stretch signal from the primary alcohol groups was exacerbated at 1190 cm 1. The increase in the FTIR spectrum of these groups in the doxorubicin and trabectedin structures of the DTP-NLC sample indicates that the nanoparticle structures are encapsulated with doxorubicin or doxorubicin and trabectedin. It is thought that this exacerbation may also come from the Trb structure since Trabectedin FTIR analysis cannot be performed.
The DSC graph of the DTP-NLC sample is given in Figure 9B. The melting temperature of the DTP-NLC structure was determined as 55,08°C in the DSC graph. A slight increase occurred in the melting temperature of the nanoparticles in the P-NLC sample with the loading of doxorubicin and trabectedin. In addition, a very similar graph was obtained to the curve obtained in the doxorubicin and trabectedin-free P-NLC sample when the DSC curve was examined. Therefore, it can be concluded from these results that the drugs are encapsulated in the lipid matrix.
The TGA graph of the DTP-NLC sample is given in Figure 9C. A large weight loss was experienced in the first stage in the range of 120°C-245°C and with a maximum temperature of 221°C as a result of the TGA analysis of the DTP-NLC structure. Another degradation occurred in the temperature range of 245°C-450°C, with a peak of 378,53°C, and it was observed that the weight loss in this step increased to -30%. The entire nanoparticle structure is degraded at temperatures after 450°C. It is thought that the decomposition behavior especially in the range of 245°C-450°C changes with the addition of drugs to the structure when compared with the P-NLC sample. The absence of one-to-one peaks of doxorubicin in the thermal gravimetric analysis curve and some change in degradation behavior in general indicate that doxorubicin or doxorubicin and trabectedin are effectively encapsulated in the structure and that the thermal stability of the drug or drugs may be increased (Trevizan et al, 2021).
The SEM image of the DTP-NLC sample is given in Figure 9D. When looked at the SEM images, it is seen that the dimensions of the DTP-NLC are in the range of 100-120 nm and in the morphology close to the sphere.
2. Formulation, characterization, and drug release studies of LAT-1 targeted and doxorubicin and trabectedin loaded nanostructured lipid carriers in gel structure
2.1 Gel formulation and characterization
The pH of DTP-NLC gel is 6,01±0,05. It was determined that the pH value of DTP-NLC charged gel was suitable for dermal use even though there was no difference between the pH values of empty carbopol gel and DTP-NLC charged gel. The viscosity value of DTP-NLC gel is 13689±101,89 cp. There is almost no difference between empty carbopol gel and DTP- NLC charged gel in terms of viscosity, and it can be said that the prepared DTP-NLC gel has an appropriate viscosity that can be applied to the skin surface in the study.
The hydrodynamic size of DTP-NLC gel is 149,60±20,93 nm and the zeta potential value is - 34,52±13,16 mV. It is thought that the dimensions may have increased slightly compared to the DTP-NLC particle group with the entry of DTP-NLC particles into the gel structure. It is expected that the zeta potential value will be negative since it is known that carbopol gel is a polyacrylic polymer, and there has been an increase in the absolute value of the negative load with the addition of minus-loaded DTP-NLC to the structure. In addition, there was an increase in the hydrodynamic size of DTP-NLC charged gel compared to DTP-NLC.
The DSC graph of the DTP-NLC-loaded gel is given in Figure 10A. It was determined considering the DSC graph that the melting temperature of DTP-NLC charged gel was 116,83°C. It is seen that it reaches a higher melting temperature compared to DTP-NLC and is in accordance with the literature (Khurana et al, 2013; Patil et al, 2015; Wen et al, 2012).
The SEM image of the DTP-NLC-loaded gel is given in Figure 10B. Structures with dimensions in the range of 160-200 nm are seen in the morphology close to the sphere when the SEM image of DTP-NLC gel is examined.
2.2 Drug release study
Time-dependent release of free doxorubicin and trabectedin solutions in pH 5 and pH 7,4 media is shown in Figures 11 A and 1 IB. The time-dependent release of doxorubicin from the DTP-NLC sample at pH 5 and pH 7,4 media is shown in Figure 12. The time-dependent release of doxorubicin from DTP-NLC gel sample in pH 5 and pH 7,4 media is shown in Figure 13. While the total amount of doxorubicin in pH 5 buffer in free form shows release in 1,5 hours, 86,38% at the 1.5th hour in pH 7,4 buffer and 100% at the 4th hour are observed. The release of trabectedin was 86% at pH 5 and 80% at pH 7,4 at the 4th hour in the free drug solution. Trabectedin appears to have a better release at pH 5 as with doxorubicin, but is thought to show a slower release as it has a lower solubility than doxorubicin. Dox HPLC analyzes were completed in the release samples; however, Trb HPLC analyses could not be presented here as they continue. It is seen according to the release data that the release of doxorubicin from the LAT-1 targeted trabectedin and doxorubicin-loaded nanostructured lipid carriers and gel formulation is much lower than that of the free form. It was also determined that the release of doxorubicin from the gel formulation was lower than its release from the nanoparticle. The acidic release of doxorubicin is higher than the physiological pH medium in both nanoparticle and gel formulation. 2% release was detected at pH 7,4 at the end of 3 hours while Dox, one of the nanoparticles, showed approximately 6% release at pH 5 at 1 hour. Dox release at 24 hours was 13% in pH 5 medium and 2,3% in pH 7.4 buffer. The release profile, which starts faster and then continues slowly, is seen. Release from the gel formulation is much slower. Doxorubicin was released at a rate of 1% after 6 hours in pH 5 buffer and 0,75% in pH 7,4 buffer. At the end of the 24th hour, Dox release of 1,61% occurred in pH 5 buffer at the end of the hour; Dox release of 1,21% occurred in pH 7,4 medium. Dox releases from nanoparticles were more abundant in the acidic medium in the literature, similar to the invention. 3. Biocompatibility Studies
3.1 Protein Binding
Table 1 shows the protein percentages that bind to NLC, P-NLC, DTP-NLC groups as a result of incubation of nanocarriers with fetal bovine serum.
Table 1. Protein binding percentages (%) at different serum: nano-carrier ratios (v:v).
Figure imgf000020_0001
In general, it was seen when DSPE-PEG-Tyrosine was bound to the NLC structure that the percentage of protein bound to the empty NLC structure decreased compared to the structure, while the percentage of protein binding increased slightly with the addition of drugs to the structures. However, higher percentages were not reached than the amount of protein bound to the NLC group without DSPE-PEG-Tyrosine. It can be thought that DSPE-PEG-Tyrosine conjugate prevents protein binding to a certain extent thanks to the PEG arm. It can be said that drugs cause an increase in protein binding because they increase hydrophobicity. The protein binding percentages of all studied ratios and nano-carrier groups are at most around ~21. It can be thought that the DTP-NLC structure is biocompatible and may remain in circulation according to the results of the protein binding experiment.
3.2 Hemolysis
No hemolysis was found for all three sample groups in the concentration range specified in all results. It can be concluded considering the literature evaluations and the protein binding data that the nanocarrier system prepared is biocompatible.

Claims

1. An effective drug delivery system in the treatment of melanoma, characterized in that it is a nanostructured lipid carrier containing doxorubicin and trabectedin which are two drugs effective in the treatment of melanoma and tyrosine ligand targeted to L- aminoacid transporter-1 (LAT-1) for the specific uptake thereof by melanoma cells.
2. A method of preparing the drug delivery system according to claim 1, characterized in that it comprises the process steps of; a) Obtaining the DSPE-PEG-Tyrosine conjugate by the esterification reaction between l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[carboxy(polyethylene glycol)-2000] (sodium salt) (DSPE-PEG-COOH) and L-Tyrosine, b) Preparing the nanostructured lipid carriers (NLC) with fat, oil, and surfactant, c) Obtaining the nanostructured lipid carriers (P-NLC) with DSPE-PEG-Tyrosine conjugate by loading the DSPE-PEG-Tyrosine conjugate into the medium during the preparation of nanostructured lipid carriers (NLC), d) Creating nanostructured lipid carriers (DTP -NLC) carrying DSPE-PEG- Tyrosine loaded with doxorubicin and trabectedin by adding doxorubicin (Dox) and trabectedin (Trb) solutions during the preparation of P-NLC.
3. A method according to claim 2, characterized in that the fat is myristic acid.
4. A method according to claim 2, characterized in that the surfactant is Tween 80.
5. A method according to claim 2, characterized in that the oil is sweet almond oil.
6. A method according to claim 2, characterized in that a gelling agent is added after step d.
7. A method according to claim 6, characterized in that the gelling agent is carbopol 940.
8. A method according to claim 2, characterized in that it comprises the process steps of; a) Dissolving 0,01-0,1 mmol L-tyrosine in 10-15 mL distilled water by dropping 100-300 pL 1-5 M hydrochloric acid (HC1) solution and heating in a magnetic stirrer, b) Adding 0,01-0,04 mmol 4-Dimethylaminopyridine (DMAP) dissolved in 0.5-2 mL of distilled water and mixing at room temperature for 30 minutes, c) Dissolving l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[carboxy(polyethylene glycol)-2000] (sodium salt) (DSPE-PEG-COOH) containing 0,005-0,02 mmol PEG in 0,5-2 mL of distilled water by means of an ultrasonic device, d) Dissolving 0,005-0,02 mmol Dicyclohexyl carbodiimide (DCC) in 0,5-2 mL distilled water :DMSO, e) Adding Dicyclohexyl carbodiimide (DCC) solution obtained in step d to DSPE-PEG-COOH solution obtained in step c and mixing on orbital mixer at 100-600 rpm at room temperature for 30 minutes, f) Adding the solution obtained in step e to the solution obtained in step b, g) Removing tyrosine, DCC and DMAP, which are not included in the compound structure, by dialyzing the DSPE-PEG-tyrosine compound obtained against distilled water for 24 hours at room temperature, h) Drying the DSPE-PEG-Tyrosine conjugate in the oven at 20-80°C after dialysis, i) Melting 20-60 mg of myristic acid and 50-100 mg of sweet almond oil at a temperature higher than the melting temperatures, j) Preparing an aqueous phase solution using 50-100 mg of tween 80 and 2-5 g of distilled water, k) Adding 1-4 mg of DSPE-PEG-Tyrosine conjugate obtained in step h to the aqueous phase and incubating at 70°C, l) Adding 1-10 mg/mL doxorubicin solution dissolved in 80-130 pL DMSO and 40-180 pg/mL trabectedin solution prepared in 80-130 pL DMSO to the lipid phase obtained in step i, m) Homogenizing by mixing at 8000 rpm for 10 minutes by means of a high speed homogenizer while slowly adding the lipid phase into the aqueous phase at 70°C, n) Dispersing with a sonicator at 70°C for 10 minutes with 10% amplitude, o) Keeping at +4°C for 24 hours for crystallization after cooling to room temperature, p) Obtaining nanostructured lipid carriers (DTP-NLC) carrying DSPE-PEG- Tyrosine loaded with doxorubicin and trabectedin.
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WO2014028487A1 (en) * 2012-08-13 2014-02-20 Massachusetts Institute Of Technology Amine-containing lipidoids and uses thereof
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