Synthesis of chimeric peptosomes hybridized with rod-shaped gold nanoparticles and aptamer-targeted for breast cancer therapy and imaging

Abstract

The invention focuses on creating chimeric peptosomes combined with gold nanoparticles and fitted with aptamers for targeted therapy and imaging of breast cancer. It aims to solve the problem of effectively targeting breast cancer cells without harming healthy cells. The method uses a special polypeptide made of polyethylene glycol (PEG) and poly(γ-benzyl L-glutamate) (PGBLG) to form a peptosome with a strong core for containing the chemotherapy drug doxorubicin. The peptosome is designed for controlled release of the drug and also allows for the inclusion of gold nanoparticles. The EpCAM aptamer is used to target cancer cells more precisely, improving therapy effectiveness and imaging through the unique properties of gold nanoparticles. This new platform enhances treatment success while minimizing side effects, representing a major advance in targeted cancer therapy.

Description

Synthesis of Chimeric Peptosomes Hybridized with Rod-Shaped Gold Nanoparticles and Aptamer-Targeted for Breast Cancer Therapy and Imaging

[0001] The innovative approach developed focuses on utilizing chimeric peptosomes, a cutting-edge combination of gold nanoparticles and aptamers, to target and treat breast cancer with precision. The peptosomes are formed from a unique polypeptide blend of polyethylene glycol (PEG) and poly(γ-benzyl L-glutamate) (PGBLG), creating a robust core capable of encapsulating the potent chemotherapy drug doxorubicin. This structure allows for controlled drug release, while also enabling the integration of gold nanoparticles for enhanced imaging capabilities.

[0002] By incorporating aptamers that specifically bind to EpCAM receptors found on breast cancer cells, the peptosomes can effectively target malignant cells while minimizing damage to healthy tissues. The inclusion of gold nanoparticles further enhances the accuracy of imaging, providing clinicians with a clearer picture of tumor localization and treatment response.

[0003] Overall, this groundbreaking technology represents a significant advancement in the field of targeted cancer therapy. By combining the precise targeting of aptamers with the therapeutic potential of gold nanoparticles and chemotherapy drugs, the invention offers a promising solution to the challenge of effectively treating breast cancer while sparing healthy cells from harm.

[0004] A61K 45 / 00 - A61K 51 / 1244

[0005] CN110237035B

[0006] Active targeting amphiphilic polypeptide nano-drug carrier and preparation and application thereof

[0007] The invention provides an active targeting amphiphilic polypeptide nano-drug carrier and preparation and application thereof, belonging to the technical field of biological medicines. The amphiphilic polypeptide takes an alkyl chain as a hydrophobic end, the polypeptide chain with an active targeting function and a side chain modified fluorescent functional molecule as a hydrophilic end, and the antitumor drug is entrapped in a hydrophobic cavity of a micelle formed by self-assembly of the amphiphilic polypeptide.

[0008] Although the above-mentioned patent is a similar attempt to provide an active nano-drug delivery product, the production process, specified purpose and ingredients are different. For instance, our solution is developed for targeting breast cancer cells and uses specified components including gold nanoparticles to achieve the final product.

[0009] US10548998B2

[0010] Multimodal silica-based nanoparticles

[0011] The present invention provides a fluorescent silica-based nanoparticle that allows for precise detection, characterization, monitoring and treatment of a disease such as cancer. The nanoparticle has a range of diameters including between about 0.1 nm and about 100 nm, between about 0.5 nm and about 50 nm, between about 1 nm and about 25 nm, between about 1 nm and about 15 nm, or between about 1 nm and about 8 nm. The nanoparticle has a fluorescent compound positioned within the nanoparticle, and has greater brightness and fluorescent quantum yield than the free fluorescent compound.

[0012] The above-mentioned invention relates to a targeted theranostic system for better and real-time detection of cancer during treatment using the attachment of various imaging agents to small-sized silica nanoparticles coated with PEG and targeted with different ligands. While our claimed patent also offers a developed formula for targeted cancer treatment purposes, the two patents have different process and components. For instance, this patent relies on silica nanoparticles while ours utilizes gold nanoparticles.

[0013] WO2020041267

[0014] GOLD NANOPARTICLE-LIGAND CONJUGATES AND METHODS OF USE

[0015] Gold nanoparticles are conjugated to phosphatidylserine-specific ligands for targeting and binding to surface-exposed phosphatidylserine on tumor cells and tumor vasculature. The ligand may be an annexin (e.g, annexin V). Tumor contrast is significantly increased using the targeted gold nanoparticles. Breast cancer tumors as small as 4 mm, for example, were detectable via computed tomography (CT) within 4 hours after injection of the conjugates, demonstrating usefulness of the conjugates as imaging agents. The targeted gold nanoparticle conjugate may further have a drug conjugated thereto that can be used therapeutically, for example, for cancer treatment. The gold nanoparticle conjugates can also be used for photothermal therapy and can be used in concert with an X-ray radiation treatment for cancer treatment.

[0016] This invention resembles our claimed one in overall purpose and the use of gold nanoparticles in cancer treatment development. However, the methods and processing techniques are different in the two patents. For instance, this one has a wider scope while ours is designed to target specified polypeptide.

[0017] IN201741038811

[0018] DEVELOPMENT OF ENGINEERED GOLD NANOPARTICLES FOR HIGH CONTRAST IMAGING OF TUMOR IN X-RAY PHOTOGRAPHY AND PHARMACOKINETIC STUDIES IN VIVO

[0019] Gold nanoparticles are synthesized and stabilized using soursop fruit extracts in a greener way. The nanoparticles were characterized using various techniques like UV-Vis spectroscopy, Transmission Electron Microscopy, Fourier Transform Infrared Spectroscopy and Zeta potentiometer. These particles are easily soluble in water, stable for more than six months and viable to breast cancer cell lines MCF-7. This study demonstrated the feasibility of gold nanoparticle based molecular cancer imaging, pharmacokinetics and photothermal therapy.

[0020] This invention also relates to targeted cancer treatment using gold nanoparticles, however, the material, methods and techniques are different compared to our design. For instance, this patent emphasizes the use of fruit extracts while ours makes no mention of this.

[0021] CN104368000

[0022] Targeting modified gold nanorod targeted drug delivery compound and application of delivery compound to anti-tumor photothermal therapy

[0023] The invention relates to a targeting modified gold nanorod targeted drug delivery compound and an application of the delivery compound to anti-tumor photothermal therapy. The delivery compound is characterized in that a two-layer lipidosome structure similar to a cell membrane structure is formed by temperature-sensitive macromolecule K3, temperature-sensitive macromolecule 09JA, lecithin and Mal-PEG-DSPE; medicines and gold nanorods are packed into the lipidosome; target molecules are modified on the surface of the lipidosome.

[0024] This invention shares certain qualities with our design as they both utilize gold nanoparticles to develop cancer treatment methods. However, their approach, formula and methods are different. For instance, ours focuses on breast cancer specifically while this one has a wider scope.

[0025] According to the studies prior art, gold rod-shaped nanoparticles have had a very effective and widespread application in the field of diagnosis and therapy, which we used as a diagnostic agent in this invention. Also, several peptomycell pharmaceutical formulations based on PEG-PGA copolypeptide have been in different phases of clinical trials, including NK-911, NK-012, NC6004 and NK105, and the results of clinical studies showed that these pharmaceutical formulations have good therapeutic efficacy and have increased the therapeutic window of many anticancer drugs. Accordingly, in this invention, a very similar form of the PEG-PGA copolypeptide (which is the unprotected form of the synthetic polypeptide PBLG) has been used to prepare vesicular nanoparticles, which are capable of simultaneously loading hydrophilic and hydrophobic agents into a nanocarrier, while micellar nanoparticles are only capable of loading hydrophobic drugs and diagnostic agents.

[0026] The invention pertains to the synthesis of chimeric peptosomes hybridized with gold nanoparticles and functionalized with aptamers for targeted therapy and imaging of breast cancer, with applications in precision drug delivery. This innovation addresses the critical challenge of lacking a therapeutic method capable of precise targeting of breast cancer cells to minimize drug-induced adverse effects on healthy cells.

[0027] The proposed solution involves the use of a dual amphiphilic polypeptide composed of polyethylene glycol (PEG) and poly(γ-benzyl L-glutamate) (PGBLG). This design creates an optimized hydrophilic core within the peptosome structure, enabling high-capacity encapsulation of the hydrophilic chemotherapeutic agent doxorubicin. The robust peptosome wall facilitates slow, controlled drug release kinetics. Concurrently, hydrophobic rod-shaped gold nanoparticles are efficiently encapsulated within the peptosome bilayer.

[0028] In this nanoformulation, the EpCAM aptamer is employed as a targeting ligand to enhance specificity toward cancer cells. The integration of aptamer-mediated targeting with the hybrid peptosome-gold nanoparticle system not only improves therapeutic precision but also enables imaging capabilities, leveraging the unique optical properties of gold nanoparticles. This multifunctional platform represents a significant advancement in targeted oncology, offering a theranostic approach to enhance treatment efficacy while reducing systemic toxicity.

[0029] Today, due to the lack of powerful diagnostic tools, we are unable to diagnose cancer early, and for this reason, this disease is considered a serious disease and a major cause of death. Over the past few decades, many efforts have been made to solve this problem. Currently, nanomedicines are being investigated as an effective solution and treatment for cancer.

[0030] Research into the development of anticancer formulations based on polymer nanoparticles for cancer treatment has also yielded promising results. The two basic problems of effective diagnosis and chemotherapy are very important in the fight against most cancers, and by using theranostic systems (systems that simultaneously carry drugs and elements required for imaging), we can hope for more effective cancer treatment.

[0031] Among the various nanoparticles, liposomal nanoparticles and some nanoparticles based on dual-acting polypeptides are in different phases of clinical trials, and some of them have entered the clinical market. A new and very suitable generation of carriers for the construction and development of theranostic systems are vesicles made of amphiphilic polypeptides called peptosomes. Studies have proven the efficiency of these types of systems for drug delivery and controlled release. These structures have the ability to simultaneously load hydrophilic and hydrophobic therapeutic and diagnostic agents in the central aqueous core and the hydrophobic bilayer membrane.

[0032] Today, a number of peptomicell nanostructures have entered different phases of clinical trials for cancer treatment, but the structural characteristics of micellar nanocarriers have limited their application in various biomedical fields, because micelles are only able to load hydrophobic drugs in their central hydrophobic core. In recent years, peptosomes have been widely used in various biomedical fields due to their advantages such as high colloidal stability, tunable hydrophobic bilayer membrane properties, and the ability to load a wide range of hydrophilic and hydrophobic drugs. Among polypeptide-based nanocarriers, PEG-b-polypeptide has been widely used in drug delivery systems due to its ease of preparation. The first generation of peptomicells, including NK-911, NK-012, NC6004, and NK105, have entered different phases of clinical trials. In peptomicell or peptosomal structures, the release of drug cargo occurs by the collapse of the nanostructure through the transfer of the hydrophilic block to the hydrophobic block, which is controlled by the secondary structures of the polypeptides. The results of the studies showed that polypeptide-based drug delivery systems have good therapeutic efficacy and have increased the therapeutic window.

[0033] The dual-peptide PEG-PGA has been approved by the US Food and Drug Administration; NC-6004 is a 30 nm peptomicell formulation prepared by self-assembly of cisplatin-conjugated PEG-PGA polypeptide in aqueous solution, which is in phase III clinical trials for the treatment of pancreatic cancer. The results showed that the tumor accumulation of NC-6004 was increased compared to albumin (90 nm) or liposomal (130 nm) nanocarriers in pancreatic tumors with dense stroma.

[0034] PBLG is the unprotected form of PGA polypeptide, and the dual-peptide PEG-PBLG can be used to construct peptosomes due to its dual-peptide properties. Therefore, in this invention, a PEG-PBLG-based peptosomal system was developed that efficiently loaded the drug doxorubicin into it. This system showed ideal physicochemical properties and controlled the release of doxorubicin. Doxorubicin is an anthracycline antibiotic and was first isolated in 1970 from a bacterial strain called Streptomyces peucetius var. caesiusin. It is routinely used alone or in combination with other drugs in the treatment of many cancers, including breast, lung, stomach, ovarian, thyroid, Hodgkin and non-Hodgkin lymphoma, multiple myeloma, sarcoma, and pediatric cancers. Nowadays, gold nanorods (GNRs) are widely used as highly effective therapeutic and diagnostic agents in theranostic systems. These nanoparticles have unique properties such as excellent optical and physicochemical properties, easy synthesis with surface modification, good chemical stability, low toxicity, high absorption capability at low dosage due to strong SPR, and tunability of absorption wavelength from visible to NIR by changing the aspect ratio of GNR nanoparticles.

[0035] Among GNR with different sizes, small-sized GNR has been widely used in biomedicine due to its advantages such as excellent scattering ability, lower toxicity, faster clearance, and tunable LSPR in the NIR region. GNR has been widely used in biomedicine due to its advantages such as strong extinction coefficient, excellent photothermal conversion efficiency, and high sensitivity for NIR imaging, OCT, and X-ray CT, and therefore has been widely used in various biomedical fields. The surfactant CTAB used in the GNR synthesis process has limited the biological application of GNR due to its high toxicity. Therefore, replacing CTAB with molecules with terminal thiols is an effective method for modifying the GNR surface. For this purpose, in this study, the ligand 11-mercaptodecanoic acid was used to reduce the toxicity of GNR nanoparticles and make them hydrophobic for loading into the peptosome bilayer membrane.

[0036] In order to target theranostic systems and for the peptosome containing hydrophobic GNR and the drug doxorubicin to reach the patient's cell or tissue, specific ligands must be embedded on their surface to specifically interact with receptors on the surface of the cell or tissue. Aptamers are short single-stranded oligonucleotides of DNA or RNA that can specifically bind to their target due to their unique three-dimensional structure. In this study, the DNA aptamer EpCAM was used, which, according to studies, is expressed more than 1000-fold in epithelial cancer tissues compared to normal epithelial cells. EpCAM is a glycosylated protein in the cell membrane with 314 amino acids that induces cell growth through upregulation of the oncogene c-myc and cyclins A / E. Due to the different expression levels of EpCAM, this protein is a good candidate for targeted drug delivery of therapeutic and diagnostic agents for the treatment and diagnosis of cancer. According to previous studies, increased expression of EpCAM protein in breast cancer causes tumor recurrence, metastatic progression and reduced survival. Also, breast, colorectal and pancreatic cancer stem cells are EpCAM positive. Therefore, in this project, by creating a theranostic system based on PEG-PBLG peptosomes containing doxorubicin in the aqueous space and hydrophobic GNR in the bilayer membrane of peptosomes and then targeting this system with EpCAM aptamer, the need of an oncologist can be met for appropriate treatment, diagnosis and follow-up of tumor growth conditions.Solution of Problem

[0037] The existing technical problem is the lack of a suitable diagnostic and therapeutic method for more precise targeting of breast cancer cells, in order to reduce drug side effects on healthy cells. New targeted drug delivery systems based on polypeptides are designed to load drug molecules in such a way that a higher amount of drug is placed inside these carriers and a lower amount of healthy cells are damaged. On the other hand, by using theranostic nanosystems that are capable of loading diagnostic and therapeutic elements into a nanocarrier, the therapeutic performance and different stages of the disease can be followed in real time. Peptosomes based on dual-layered polypeptides provide a suitable space for simultaneous loading of different diagnostic and therapeutic agents by creating bilayer vesicles. Depending on the type of dual-layered polypeptide used in the structure of these types of nanocarriers, different pharmaceutical and diagnostic agents can be used.

[0038] In this invention, in the first phase, the hydrophobic polypeptide PBLG was synthesized using ring-opening polymerization of the monomer gamma-benzyl-n-carboxy-anhydride (BLG-NCA) in the presence of the initiator n-hexylamine (n-hex) and then attached to PEG using EDC / NHS chemistry. Then, the amphiphilic polypeptide PEG-PBLG was converted into peptosome using the double emulsion method and the simultaneous loading of the hydrophobic drug DOX and GNR was performed in the aqueous central core and the hydrophobic bilayer membrane of the peptosome with high efficiency. Finally, in order to prepare a targeted formulation, the outer surface of the peptosome was conjugated with the EpCAM aptamer.

[0039] 1) Structure and various parts of the invention:

[0040] First, the hydrophobic polypeptide PBLG was synthesized using ring-opening polymerization by the following method. For this step of the synthesis, 0.789 g of BLG-NCA monomer was placed in a 50 ml double-necked flask and dissolved in 894.7 ml of dry DMF, and then the n-hex initiator solution (3.3 μL of n-hex in 189 μL of dry DMF) was added dropwise to the monomer solution and rotated under argon for 72 hours at 25 °C. Then, precipitation was carried out with 20 ml of diethyl ether antisolvent, and this process was repeated 3 times. Subsequently, the resulting white precipitate was freeze-dried in a freeze-dryer, and the obtained polymer was stored at 4 °C until use. Next, for the synthesis of the amphiphilic polypeptide using EDC / NHS chemistry, the terminal amine of PBLG was covalently attached to the carboxylic acid group of PEG. In this step, 100 mg of PEG was dissolved in 5 ml of dry DMF, then 1.53 mg of EDC and 0.920 mg of NHS were added and the mixture was stirred at room temperature for 2 hours to activate the carboxylic acid group of PEG. Then 290 mg of PBLG was added to the previous solution and stirred at room temperature for 24 hours. Finally, the obtained amphiphilic polypeptide was precipitated in 15 ml of diethyl ether and washed three times with a 70:30 v / v solution of diethyl ether / methanol to remove excess EDC / NHS. Then, the resulting diblock copolymer was dried by freeze-drying and stored at 4 °C until use.

[0041] 2) Determination of structural properties of PBLG and PEG-PBLG diblock

[0042] After the synthesis of PBLG, the structural properties of the polypeptide were confirmed by nuclear magnetic resonance (1H-NMR) at room temperature and in CDCl3 solvent using a Bruker Avance 300 MHz NMR spectrometer [Pic. 1]. Also, the molecular mass of the polymer and the molecular mass distribution of the polymer were measured using GPC analysis (Shimadzu LC-20Ah with StyragelHR2 column) in THF / DMF solvent with a ratio of 1:1 (Table 1). Also, after the synthesis of the PEG-PBLG amphiphilic polypeptide, the successful connection of the two PEG and PBLG blocks was confirmed using HNMR, 13CNMR and FTIR analyses [Pic. 2] and [Pic. 3]. On the other hand, the thermal properties of the PEG, PBLG and PEG-PBLG polymers were investigated by DSC and TGA analyses [Pic. 4].

[0043] 3) Synthesis of hydrophobic gold rod nanoparticles (MUA.GNR)

[0044] Small-sized GNR were prepared using the seedless method that has been synthesized in previous studies. In this method, 0.2 M (0.092 g) of cetyltrimethylammonium bromide was placed in a 100 mL round-bottom flask and placed in 15 mL of gently rotating deionized water at 25°C until completely dissolved and a clear solution was obtained. Then, 15 mL of HAuCl4.3H2O solution with a concentration of 1 mM was added to the CTAB-containing flask solution, and after 10 minutes, silver nitrate solutions (900 μL, 4 mM), HCL (37%, 36 μL), and ascorbic acid (225 μL, 85.8 mM) were added, respectively. After the solution became colorless, the magnet was removed from the flask and 22.5 microliters of cold sodium borohydrate solution with a concentration of 0.1 molar was prepared and added to the flask and the reaction was left overnight at 25oC without rotation. The dark red color of the solution indicates the synthesis of GNR nanoparticles. Finally, the synthesized GNR was centrifuged (20 minutes at 15,000 rpm) and washed three times with deionized water to remove excess CTAB from the structure of the gold nanoparticles. Hydrophobic GNR nanoparticles (MUA.GNR) were synthesized using the ligand exchange method. At this stage of the synthesis, a solution of 11-mercaptodecanoic acid in ethanol (1 mL, 0.02 mM) was added dropwise to the GNR solution (5 mL, 20 nM) and left overnight at room temperature with a slow rotation of the stirrer. Then, the excess ligand was washed with 500 μL of chloroform. Finally, the GNR.MUA nanoparticles were centrifuged (20 min, 1500 rpm) and the supernatant was removed, and the resulting plate was dispersed in 1 mL of THF.

[0045] 4) Physicochemical properties of MUA.GNR

[0046] After the synthesis of GNR and MUA.GNR nanoparticles, their structure was investigated by UV spectroscopy [Pic. 5] part A. In the next step, the morphology of MUA.GNR was examined using transmission electron microscopy using a LEO 912 TEM device at a voltage of 80 kW TEM imaging [Pic. 5] part B.

[0047] 5) Preparation of DOX and MUA.GNR loaded peptosome formulation

[0048] DOX / GNR.MUA-loaded peptosome was prepared by double emulsion method, and the resulting formulation was named Pep@MUA.GNR-DOX. In the first step, 5 mg of the PEG-PBLG dual polypeptide was dissolved in 800 μL of dichloromethane solvent, and then 200 μL of GNR.MUA solution was added to it (S1) and probe-sonication was performed. Then, 100 μL of 2 mg / mL DOX solution was added dropwise to S1 solution and probe-sonication was performed for 5 min, at which point an emulsion solution was formed (E1). Next, E1 was added dropwise to 4 mL of 0.5% PVA solution in PBS and probe-sonication was performed for 15 min in an ice bath (E2). Next, E2 was added dropwise to 10 ml of 0.1% PVA solution in PBS and left overnight at room temperature while rotating at 800 rpm. Finally, to remove free DOX and excess PVA, the Pep@MUA.GNR-DOX formulation was washed with 2 ml of deionized water and centrifuged at 4 °C at 15,000 rpm for 20 minutes. The size, particle size distribution and surface charge of the formulations are given in Table 2.

[0049] 6) Determination of DOX and MUA.GNR loading

[0050] The amount of DOX loaded in the peptosome nanoparticles was measured indirectly by measuring the amount of free DOX in the supernatant using UV spectroscopy at a wavelength of 480 nm. In order to calculate the concentrations obtained from the adsorption, standard solutions of DOX were prepared in PBS and the amount of free drug in the supernatant was calculated and multiplied by the final volume of the supernatant. Then, the percentage of drug loading capacity and drug loading efficiency were calculated using the following formulas.

[0051] Percentage of drug loading capacity = (initial amount of drug in the formulation - amount of free drug in the supernatant / initial amount of drug in the formulation) × 100

[0052] Percentage of drug loading efficiency = (amount of drug loaded in the peptosome / amount of polypeptide used in the formulation) × 100

[0053] In this invention, the loading rate of GNR.MUA was determined using ICP-OES.

[0054] 7) DOX release profile from Pep@MUA.GNR-DOX

[0055] The release profile of DOX was investigated in three environments including phosphate buffer with pH = 7.4, PBS with 30% of FBS and citrate buffer with pH = 5.4. In this study, 1.5 ml of the solution containing Pep@MUA.GNR-DOX formulation in drug release medium buffer with a concentration of 500 μg / ml of DOX was poured into a dialysis bag with MW cut off = 6-8 kDa and then placed in a container containing 20 ml of citrate buffer or PBS. The drug release study was performed in a shaking incubator at 37°C with a rotation speed of 70 rpm, and at certain time intervals (1, 2, 3, 4, 5, 24, 48, 72, 96, 120, 144, 168, 192, 216 and 240 hours) 1 ml of the buffer was removed from the container and replaced with 1 ml of the studied medium buffer. Finally, the amount of DOX released in each sample was measured using fluorescence spectroscopy at excitation and emission wavelengths of 480 and 580 nm, respectively [Pic. 6].

[0056] 8) Peptosome colloidal stability in vitro

[0057] The serum stability of Apt-Pep@MUA-GNR.DOX and Pep@MUA-GNR.DOX was investigated in PBS medium containing 10% FBS for 48 hours. In this study, 1 ml of targeted and non-targeted peptosomes solution was placed in PBS buffer containing 10% FBS in a shaker incubator at 37 °C. Subsequently, the particle size and size distribution were investigated using DLS analysis for 48 hours. The shelf life of targeted and non-targeted formulations was also studied for 6 months at 4°C [Pic. 7].

[0058] 9) Conjugation of EpCAM aptamer on the surface of peptosome nanoparticles

[0059] In this study, EpCAM aptamer with a terminal thiol functional group was covalently attached to the surface of Mal-PEG-PBLG through a thiol-maleimide reaction. In order to prepare the targeted formulation, after preparing Pep@MUA-GNR.DOX using the double emulsion method, a concentration of 5 mg / mL of this formulation was dissolved in nuclease-free water, and then 20 μL of 10 μM EpCAM aptamer was added to it and kept in the dark at 4°C for one day with gentle rotation. Finally, in order to remove free aptamer, it was centrifuged at 15,000 rpm for 30 minutes at 4°C, and the amount of free aptamer in the supernatant was examined using a Nanodrop spectrophotometer.

[0060] 10) Physicochemical properties of peptosomal nanoformulations

[0061] After preparing different peptosomal formulations including Blank Pep, Pep@MUA-GNR, Pep@MUA-GNR.DOX and Apt-Pep@MUA-GNR.DOX at a concentration of 5 mg / ml in deionized water, 100 μl of them were diluted with 900 μl of deionized water and the particle size and their dispersion index and the surface electric potential of the nanoparticles were measured using the dynamic light scattering technique of the Zeta sizer device at a wavelength of 611 nm and a temperature of 45 ° C under the conditions of 4mW Ne.He laser 9o Scattering angle (Table 2). Also, the surface electric potential of the nanoparticles was read at a temperature of 45 ° C. AFM and FESEM were used to determine the morphology and particle size distribution of peptosome nanoparticles.

[0062] 11) Study of the efficacy of peptosomal nanoformulations in vitro by MTT test and flow cytometry

[0063] Evaluation of the cellular uptake of Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX formulations was performed by flow cytometry on 4T1 and CHO cell lines. In this study, 800 μl of complete culture medium containing 104×6 cells was cultured in a 24-well plate and 24 hours were given for the cells to adhere to the bottom of the plate and grow. In the next step, the serum-containing culture medium was replaced with a culture medium containing targeted, non-targeted nanopeptosomes and free DOX at a specific concentration (600 μl, 5 μg / ml) and incubated for 2 hours in the dark in an incubator. After the incubation time, the culture medium was removed from the cells and the cells were washed three times with cold PBS with pH = 7.4. In the next step, trypsin enzyme was used to separate the cells from the bottom of the plate and the cell suspension in complete culture medium was separated from each well of the plate and centrifuged at 4°C at 1400 rpm for 7 minutes. Then, the supernatant was removed and the cell plate was washed with cold PBS and this was repeated twice. Finally, the cell plate was resuspended in 200 μl of cold PBS and read by a FACSCalibur BD device in Channel FL2. The obtained data were then analyzed by Flow JO software [Pic. 8].

[0064] The in vitro cytotoxicity of free DOX, Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX with equal concentrations of DOX in cancer cells (4T1 and MCF-7) and normal cells (CHO) was investigated using the MTT assay [Pic. 9]. For this purpose, 4T1, MCF-7 and CHO cell lines were cultured in 96-well plates with 100 μl of complete culture medium containing 5000 cells per well. After 24 hours when the cells adhered to the bottom of the plate and grew, the cell culture medium was replaced with culture medium containing DOX drug at different concentrations (20 to 0.312 μg / ml) with 4 replicates for each concentration and incubated for 6 hours in an incubator at 37°C. After 6 hours, the culture medium containing the drug was replaced with fresh culture medium and placed in an incubator at 37°C for 48 hours. In the next step, 20 μL of MTT dye solution with a concentration of 5 mg / mL was added to the cell culture medium and incubated for 4 hours at 37°C. Subsequently, the culture medium containing the MTT dye was removed and 100 μL of dimethyl sulfoxide solvent was added to each well to dissolve the cells and formazan crystals. In the next step, the plate was placed on a shaker at 6000 rpm for 5 minutes and its absorbance was read by a microplate reader at a wavelength of 570 versus 630 nm. The viability of the control group cells to which no drug was added was considered 100% and the percentage of cell survival was calculated by the following formula.

[0065] Cell survival percentage (%) = (absorption per well / average absorption of control wells) × 100

[0066] 12) Study of anticancer efficacy of peptosomal nanoformulations in vivo

[0067] In this invention, formulations prepared from female BALB / c mice were used to investigate the in vivo antitumor efficacy. In this study, in order to induce tumors in mice, 80 μl of 4T1 cell suspension with a density of 105×4 in sterile PBS was injected subcutaneously into mice weighing 14 to 22 g. After a week, when the tumor volume of the mice reached 20-30 mm3, the mice were divided into 5 groups of 5 and 5 mice were placed in each group. Free DOX, Pep@MUA.GNR-DOX, Apt-Pep@MUA.GNR-DOX and Pep@MUA.GNR were injected intravenously into the tail of the mice at a dose of 5 mg / kg. and tumor volume, mouse weight, and mouse survival were studied for 30 days [Pic. 10].

[0068] 13) Study of tissue distribution and tumor penetration (in vivo)

[0069] In BALB / c mice bearing 4T1 tumors with a tumor size of about 200mm3, Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX formulations were injected at a dose of 5 mg / kg via the tail vein. Then, the mice were euthanized at different hours after injection (6 and 24 hours) and their main organs including heart, kidney, liver, spleen, lung and tumor were separated and washed three times with PBS solution. . The DOX signal level in each organ was measured using an animal fluorescence imaging device (KODAK IS in vivo imaging system). The results obtained show that at both times after injection, the targeted formulation had a higher accumulation in the tumor tissue. In the next step, region of interest (ROI) analysis was performed for tumor tissue and other organs using KODAK Molecular Imaging software 5.0. The results show that the mean intensity of DOX fluorescence signal of Apt-Pep@MUA.GNR-DOX formulation at the tumor site is significantly higher than that of Apt-Pep@MUA.GNR-DOX (**** p≤0.0001).

[0070] 14) CT scan imaging in vivo

[0071] Balb / c mice bearing 4T1 tumor with a tumor size of about 200mm3 were injected with Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX formulations at a dose of 5 mg / kg via the tail vein. Then, the mice were anesthetized at different times after injection (6 and 24 hours) and CT scans were taken of the tumor site. The results of the CT scans showed that the signal intensity of the CT scans of the tumor tissue of the mice that received the targeted nanopeptosome formulation was higher compared to the non-targeted type, and also that the intensity of this signal was higher in the mice that received the non-targeted nanopeptosome formulation compared to the control group.Advantage Effects of the Invention

[0072] • Reducing the side effects and systemic toxicity of doxorubicin

[0073] • Ability to take pictures simultaneously during the patient's drug treatment and follow the treatment process

[0074] • Ability to take dual imaging of the formulation and determine the exact location of the tumor tissue

[0075] • Use of biocompatible and biodegradable polypeptide in manufacturing peptosomes

[0076] • Low cost of manufacturing the formulation

[0077] • Highly effective targeted treatment method

[0078] • Minimized risks in nao drug delivery

[0079] Shows a general flowchart of the developed solution.

[0080] Shows a general flowchart of the production process for the developed product.Examples

[0081] The implementation method is as follows: first, the hydrophobic polypeptide PBLG was synthesized using the ring-opening polymerization technique. In the next step, PBLG was attached to PEG using EDC / NHS chemistry, thus obtaining the dual-drug polypeptide PEG-PBLG. Then, the stated formulation was prepared by the double emulsion method and a certain amount of the drug doxorubicin and the synthesized hydrophobic rod gold nanoparticles were loaded into peptosomes composed of the dual-drug polypeptide PEG-PBLG. After preparing the peptosomal nanoparticles, the resulting precipitate (5 mg NPs) was dispersed in 1 ml of nuclease-free water and esterified with 20 μl of a stock solution with a concentration of 10 μM EpCAM aptamer at 4 °C for one day. Finally, the resulting product is centrifuged at 15,000 g for 20 minutes to obtain particles bound to the aptamer Apt-Pep@MUA.GNR-DOX. Then, the dosage is determined based on the patient's need for the drug.

[0082] Here are some of the charts and images referenced in the solution which contain visual and numerical data:

[0083] [Pic. 1] An HNMR spectrum of synthetic PBLG

[0084] [Pic. 1]

[0085]

[0086] [Pic. 2] A: 1HNMR spectrum of PEG-PBLG copolymer, B: 13CNMR spectrum of PBLG (red), PBLG (blue) and PEG-PBLG (green)

[0087] [Pic. 2]

[0088]

[0089] [Pic. 3] FTIR spectra of (a) PBLG, (b) Mal-PEG-COOH and (c) PEG-PBLG

[0090] [Pic. 3]

[0091]

[0092] [Pic. 4] A: DSC of PEG (red), PBLG (blue) and PEG-PBLG (black). B: TGA of PEG (green), PBLG (red) and PEG-PBLG (blue).

[0093] [Pic. 4]

[0094]

[0095] [Pic. 5] UV spectroscopy for (A) GNR and MUA.GNR, (B) TEM image for MUA.GNR

[0096] [Pic. 5]

[0097]

[0098] [Pic. 6] DOX drug release from Pep@MUA.GNR-DOX in PBS, PBS with 30% v / v FBS and citrate buffer

[0099] [Pic. 6]

[0100]

[0101] [Pic. 7] Serum stability of Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX in PBS with 10% FBS after 1 and 2 days of storage in shaker incubator at 37°C

[0102] [Pic. 7]

[0103]

[0104] [Pic. 8] Flow cytometric analysis to investigate the uptake of DOX in CHO, 4T1 cell lines after 2 hours of exposure to free DOX, Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX

[0105] [Pic. 8]

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[0107] [Pic. 9] MTT assay for (A) 4T1, (B) MCF-7 and (C) CHO cell lines after 48 hours of exposure to free DOX, Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX

[0108] [Pic. 9]

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[0110] [Pic. 10] Study investigating the therapeutic efficacy of peptosomal nanoformulations after their intravenous administration of 5 mg / kg for one month

[0111] [Pic. 10]

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[0113] [Table 1] Results of GPC analysis for PBLG polypeptide

[0114]

[0115] [Table 2] Size, PDI and surface charge of different types of peptosome nanoformulations

[0116]

[0117] This invention presents a solution that can easily and at the lowest cost manufacture a theranostic and targeted drug delivery system against breast cancer with fewer side effects. By minimizing negative impacts including cardiac complications, and the ability to take simultaneous images in the pharmaceutical industry, this claimed solution achieves targeted drug production and can be used in the clinic to treat breast cancer. Health care facilities, hospitals, clinics, and companies that specialize in nanomedicine and cancer treatment would benefit greatly from the proposed patent.

Claims

A method for synthesizing chimeric peptosomes hybridized with rod-Shaped gold nanoparticles and aptamer-targeted is designed for breast cancer therapy and imaging with minimized side effects.According to claim 1, the claimed invention is an EpCAM aptamer-targeted theranostic system, based on PEG-PBLG copolypeptide-based vesicular nanoparticles loaded with the drug doxorubicin as a therapeutic agent and hydrophobic gold rod nanoparticles as a CT imaging agent.According to claim 1, the method starts with synthetizing a hydrophobic polypeptide polygammabenzyl L-glutamate (PBLG) with a desired molecular mass by a ring-opening polymerization method by changing the ratio of the monomer polygammabenzyl L-glutamate N-carboxyanhydride to the initiator N-hexylamine. This synthetic polymer has a molecular weight of 14962 g / mol and a uniform molecular mass distribution (1.19).According to claim 1, the developed product has a size of 165 nm and a dispersion index of 0.091, the size of the designed peptosome nanoparticles is less than 200 nm and has a very uniform particle size distribution, which is very suitable for intravenous injection for effective cancer treatment and diagnosis.According to claim 1, the loading rate of doxorubicin (%EE) in this formulation is 3.6±42%.According to claim 1, cytotoxicity tests were performed using the MTT assay method on EpCAM-positive breast cancer cell lines of the human model (MCF-7), the mouse model (4T1) and the EpCAM-negative normal cell (CHO). The results of the MTT cytotoxicity test indicated excellent toxicity of both targeted and non-targeted formulations, and the cytotoxicity of the targeted formulation was statistically significantly higher than that of the non-targeted formulation in EpCAM-positive cell lines (MCF-7 and 4T1). However, no significant difference was observed in the toxicity of the targeted and non-targeted formulations in the EpCAM-negative cell line (CHO).According to claim 1, the cellular uptake test by flow cytometry showed that the cellular uptake of doxorubicin loaded in the targeted formulation (Apt-Pep@MUA.GNR-DOX) in the EpCAM-positive cell line (4T1) was higher than that of the non-targeted formulation (Pep@MUA.GNR-DOX). On the other hand, the cellular uptake of the targeted and non-targeted formulations in the EpCAM-negative cell line (CHO) was the same.According to claim 1, in order to understand the distribution of the formulations in the body and the extent of their tumor accumulation, in vitro fluorescence imaging was performed 6 and 24 hours after injection of the targeted and non-targeted formulations. The results obtained show that the average intensity of the fluorescent signal of the targeted formulation at the tumor site is significantly higher than that of the non-targeted formulation (**** p≤0.0001).According to claim 1, in an animal model that was tumorized subcutaneously with 4T1 cells, it was used intravenously and a significant reduction in tumor size was observed compared to the group treated with the free drug along with a decrease in mortality.According to claim 1, in vivo CT scan images of tumor tissue of this invention showed that the intensity of CT scan signal after 6 and 24 hours from the time of injection of the targeted formulation with aptamer in tumor tissue is higher than that of the non-targeted formulation.According to claim 1, after 30 days from the injection of the synthesized formulation, cytotoxicity was evaluated using hematoxylin-eosin (H&E) staining of tissues in vital organs and tumor tissue.According to claim 1, the evaluation showed less cardiac damage of the synthesized formulation compared to the free drug and more extensive necrosis of tumor tissue of mice treated with the targeted and non-targeted formulations.

Images