Nanodrug product with controlled release of HOCL at tumor site for lung cancer treatment

Abstract

This invention introduces a method of development for a nanodrug that releases HOCl at tumor sites for treating lung cancer. The process involves creating alginate chitosan nanoparticles, measuring folate conjugation to chitosan, studying the properties of the nanoparticles, assessing HOCl release and cytotoxicity, and evaluating the impact on cancer cells by analyzing protein expression. Additionally, the penetration and effects of the nanoparticles are studied in a mouse model of lung cancer to understand their behavior in vivo. Overall, this patent aims to develop a targeted and effective treatment for lung cancer with minimal side effects on healthy tissues.

Description

Nanodrug Product with Controlled Release of HOCl at Tumor Site for Lung Cancer Treatment

[0001] HOCl has been studied as a potential agent for cancer treatment in recent years and has been shown to have cytotoxic effects on cancer cells and induce oxidative stress, apoptosis, and autophagy. It can also modulate the expression of some proteins involved in cancer growth and survival.

[0002] While HOCl shows promising anticancer effects, its clinical application faces many challenges, including its stability, bioavailability, and targeted delivery. The drug delivery system designed in the present invention provides a solution for the generation of HOCl at the tumor site, which leads to controlled release, increased drug stability, and targeted delivery to cancer cells. Targeted drug delivery of hypochlorous acid to lung cancer tissue is achieved using perfluoropentane (PFP)-coated alginate nanoparticles with folic acid-conjugated chitosan coating.

[0003] This patent presents a design of a suitable nanodrug for pulmonary and targeted delivery of hypochlorous acid to tumor sites in lung cancer patients. It also provides a method for sustained production of hypochlorous acid at the cancer site which ensures control and regulation of drug release and increases drug delivery efficiency.

[0004] A61K 33 / 00 – A61P 39 / 06 – B82Y 5 / 00

[0005] CN108578388

[0006] SELF-ASSEMBLED NANO TARGETED CARRIER DRUG FOR PRECISELY CURING LUNG CANCER AND PREPARING METHOD THEREOF

[0007] The invention relates to the field of medical treatment and discloses a self-assembled nano targeted carrier drug for precisely curing lung cancer and a preparing method thereof. The preparing methodcomprises the following preparing processes that 1, polypeptide powder is dissolved in deionized water, so that a polypeptide solution is prepared; 2, chitosan and erlotinib hydrochloride are added to the polypeptide solution, so that a pregelatinized gel is prepared; 3, the pregelatinized gel is frozen and smashed to a nano grade, a saline solution is added to the pregelatinized gel so that the pregelatinized gel can be gelatinized, the gelatinized gel is frozen and dried, and thus self-assembled nano particles are prepared, namely the nano targeted carrier drug for precisely curing lung cancer.

[0008] This mentioned invention is similar to our claimed design in promising a targeted treatment for lung cancer which involves nanoparticles and controlled release. However, the composition here includes different elements and the process is different. Therefore, the two patents are alike in overall purpose but their function and design is different, for instance, this solution, unlike ours, does not rely on hypochlorous acid.

[0009] IN201641020907

[0010] NANOPARTICLE BASED DRUG DELIVERY SYSTEM TO TARGET CANCER CELLS

[0011] The present invention is in the technical field of nanoparticle delivery system, in particular, a self-assembling reconstituted high density lipoprotein complex comprising a combination of: a pH-specific polymer, a cross linker, a receptor binding component (R), and a drug (D); wherein the pH-specific polymer crosslinked to receptor binding component (R) and Drug (D), encapsulated within the nano matrix, for targeted and controlled release of drug for cancer treatment.

[0012] This mentioned patent resembles our design in general function of offering NP drug delivery with controlled release for cancer treatment. But the components and methods of the two patents vary, for example, this one does not rely on hypochlorous acid in their solution like ours. Moreover, this system has a broader scope while ours focuses primarily on lung cancer treatment via pulmonary drug administration.

[0013] CN110251689

[0014] CHITOSAN NANO MATERIAL FOR TREATING LUNG CANCER AND PREPARATION METHOD THEREOF

[0015] The invention discloses a chitosan nano material (erlotinib-quaternary ammonium salt-heptamethine cyanine dye-chitosan, CE7Q) for treating lung cancer and a preparation method thereof. The nano material is the CE7Q nano material formed by modifying a molecular targeting drug erlotinib, quaternary ammonium salt and a heptamethine cyanine dye on a chitosan surface. The CE7Q nano material has both molecular targeting and better photothermal conversion efficiency, and achieves the combination of photothermal therapy and molecular targeted therapy to improve the sensitivity of lung cancer resistant cells to erlotinib.

[0016] The above mentioned patent provides a solution for efficient drug delivery using nano material specifically for lung cancer, so it shares certain qualities with our claime done. However, the methods, process and focus of the two designs differ, for example, this one includes photothermal therapy while ours does not. Also our solution utilizes hypochlorous acid while this one makes no mention of it.

[0017] WO2017180815A1

[0018] Treatment of cancer with hypochlorous acid

[0019] The present invention relates to hypochlorous acid compositions and their therapy for cancer patients.

[0020] This mentioned invention shares common ground with our claimed one in utilizing hypochlorous acid to develop cancer treatment methods. However, unlike our design, this patent includes various embodiments with a wide range of application in the treatment of many forms of cancer. Whereas our developed solution focuses on targeted drug delivery and controlled release specifically for lung cancer. Moreover, this one features mechanisms and processes different from ours, for example, It does not specifically involve PFP chitosan alginate nanoparticles fabrication or the use of ultrasound in HOCl release profile investigation.

[0021] This patent involves the development of a nanodrug product with controlled release of HOCl at tumor site for lung cancer treatment. The first step of the process includes the fabrication of alginate chitosan nanoparticles through nano-emulsion and ionic gelation procedure. Then the folate conjugated to chitosan is measured using fluorometry, allowing researchers to accurately determine the amount of the targeting agent present in the nanoparticles. This step is essential for ensuring targeted delivery of the nanodrug to cancer cells in the lungs, minimizing potential side effects on healthy tissues.

[0022] Next is the investigation of the properties of HOCl-PFP alginate chitosan nanoparticles which involves a detailed morphological study to understand their structure and stability. The measurement of HOCl release from the nanoparticles and the assessment of cytotoxicity using the MTT method provide valuable insights into the efficacy and safety of the nanodrug. Furthermore, Western blot analysis is utilized to evaluate the expression of proteins involved in triggering apoptosis in cancer cells.

[0023] Lastly, the penetration and effect of the nanoparticles are studied in an orthotopic murine model of lung cancer, providing crucial information on their in vivo behavior and potential clinical application.

[0024] Lung cancer accounts for approximately 14% of all new cancer cases reported worldwide. In 2022, lung cancer caused an estimated 2.09 million deaths worldwide.

[0025] In general, there are 2 main types of lung cancer that have different growth and spread patterns. 1) Non-small cell lung carcinoma (NSCLC), which accounts for 85% of lung cancer cases, and 2) small cell lung carcinoma (SCLC), which accounts for the rest. Unfortunately, early detection of lung cancer remains challenging, and approximately 50% of lung cancer cases are metastatic at the time of diagnosis, significantly reducing the survival rate to only 10% at five years.

[0026] Metastatic lung cancer is a stage of lung cancer in which malignant cells can break away from the tumor and travel to other areas of the body through the blood or lymphatic system. Non-small cell lung cancer (NSCLC) has a higher tendency to metastasize to the brain compared to small cell lung cancer (SCLC). It is estimated that 30-50% of patients with NSCLC and 10-20% of patients with SCLC may develop brain metastases during the course of their disease. Brain metastases can cause a variety of symptoms, including headaches, seizures, neurological deficits, and changes in cognitive function. Management of brain metastases often involves a combination of treatments, such as surgery, radiation therapy, and in some cases, systemic therapies.

[0027] In non-small cell lung cancer (NSCLC), the most common metastatic sites are bone, followed by other parts of the lungs, brain, liver, adrenal glands, and distant lymph nodes. In small cell lung cancer (SCLC), the four most common sites of metastasis at diagnosis are liver, bone, brain, and other parts of the lung.

[0028] In non-metastatic lung cancers, resection of the lesion serves as the primary treatment approach. However, this procedure is only performed in a fraction of NSCLC patients, typically between 10% and 20%, depending on factors such as the size of the lesion, its location, and the patient's overall health and respiratory status. Chemotherapy is often used as the primary treatment for lung cancer when surgery is not an option.

[0029] The inherent genetic diversity within lung cancer subtypes adds a layer of complexity to the design of treatment strategies. Different types of lung cancer are associated with different genetic mutations, which makes it necessary to design treatment strategies for each subtype individually. In general, treatment strategies include surgery, chemotherapy, radiotherapy, and targeted therapies. The use of chemotherapy and radiotherapy to treat lung cancer is usually associated with problems, including side effects and reduced quality of life. In addition, genetic diversity within lung cancer subtypes can make it difficult to design targeted therapies for each patient individually. Consequently, further research in the identification of target molecules and the design of targeted therapies for each lung cancer subtype is of great importance.

[0030] It is also essential for anticancer drugs to effectively penetrate tumor tissue and achieve the concentration necessary for optimal tumor eradication. Inadequate drug concentration at the tumor site often leads to lower antitumor efficacy and concerns about drug resistance. Intravenous administration also results in widespread distribution of the drug to various organs, resulting in suboptimal drug levels at tumor sites. As a result, high doses are necessary to achieve therapeutically effective concentrations. Unfortunately, such high doses often result in severe side effects, especially in tissues such as hair, skin, spleen, and liver that have a high proliferation rate.

[0031] Therefore, there is an urgent demand for innovative therapies that can enhance the safety and efficacy of treatment, overcome existing limitations, and dramatically improve the treatment outcomes of patients with lung cancer.

[0032] - Pulmonary drug delivery

[0033] Intravenous (IV) administration of drugs for the treatment of lung cancer is ineffective because the bronchial artery is the main source of blood supply to lung cancer, which accounts for only 7% of cardiac output. Therefore, intravenous administration results in inadequate drug accumulation in tumors and systemic adverse reactions in normal and healthy tissues.

[0034] Pulmonary drug delivery results in effective drug delivery to the lungs and is often administered via oral or nasal inhalation. In this approach, anticancer drugs are delivered directly to the site, ensuring high drug concentrations within tumors. Pulmonary drug delivery increases drug accumulation and retention, and the half-life of the drug in the tumor, and by preventing the drug from entering the blood, it reduces its potential toxicity in normal and healthy tissues. For the treatment of lung cancer, inhalation therapy is a promising approach for the treatment of lung cancer, with significantly improved local drug distribution, increased drug accumulation in the lungs, and reduced systemic drug distribution, thereby reducing associated toxicities. In addition, a significant fraction of inhaled drugs is detected in lymph nodes, which suggests an advantage in the treatment of lung cancer that has metastasized to these nodes. The absorption of drugs into the lymphatic circulation makes their redistribution to the peripheral airways beneficial, especially in cases of lung cancer metastasis. In the ongoing effort to improve existing treatments for lung cancer, the design of a drug delivery system that can be used via inhalation is of interest.

[0035] - Hypochlorous acid (HOCl)

[0036] Hypochlorous acid (HOCl) is one of the main compounds in the regulation of the immune system and acts as an immunostimulatory agent. This means that this compound has the ability to increase the immune system's response to foreign agents. In addition, HOCl has the ability to facilitate apoptosis, a form of programmed cell death, which is essential in inhibiting the growth of harmful cells such as neoplastic cells. This molecule is produced by certain leukocytes as part of the immune response against pathogens. Notably, neutrophils, as a subset of leukocytes, release large amounts of HOCl when they encounter microbial antagonists.

[0037] Hypochlorous acid (HOCl) is also produced in living organisms through a biochemical reaction. This compound is formed when hydrogen peroxide reacts with chloride ions under the catalysis of the enzyme myeloperoxidase (MPO) (a co-peroxidase released by activated neutrophils). This chemical reaction results in the formation of HOCl and its corresponding anion, -OCl (hypochlorite). HOCl exhibits a remarkable ability to destroy pathogenic cellular structures, ultimately leading to their destruction. Its strong reactivity with biochemical substrates, including proteins, lipids, and nucleic acids, is crucial for its function in innate immunity, microbial clearance, and even anti-inflammatory therapeutics. Recent scientific research has shown promising results for HOCl as a treatment for lung cancer.

[0038] - Mechanism of Hypochlorous Acid (HOCl)

[0039] Oxidative stress: A condition that occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to counteract or detoxify their harmful effects through neutralization by antioxidants. HOCl is a highly reactive compound and has a unique capacity to induce oxidative stress within cells. In the case of cancer cells, oxidative stress causes significant damage to cellular proteins, lipids, and DNA. DNA damage and oxidative stress induced by HOCl trigger diverse apoptotic cascades in cancer cells. These cascades involve a complex series of molecular events that lead to the activation of caspases (a family of enzymes that initiate apoptosis, or programmed cell death). The PI3K / Akt pathway is a signaling pathway that promotes survival and growth in response to extracellular signals. The NF-kB pathway is a protein complex that controls DNA transcription, cytokine production, and cell survival. Both of these pathways are critical for cell survival. By disrupting these critical pathways, HOCl leads to an imbalance in cellular processes and drives cancer cells toward apoptosis.

[0040] In the cell membrane, NADPH oxidase (NOX) generates extracellular superoxide anions either spontaneously or by the enzyme superoxide dismutase (SOD).

[0041] Exogenous HOCl (in pharmaceutical form) reacts with superoxide anions present in cell membranes to produce hydroxyl radicals, which induce lipid peroxidation (LPO) (Lipid peroxidation is a chain of reactions involving the oxidative damage of lipids that occurs when free radicals steal electrons from cell membrane lipids, resulting in cell damage.) This in turn induces apoptosis. Low concentrations of HOCl can effectively react with hydrogen peroxide to produce singlet oxygen: HOCl + H2O2 → 1O2 + H2O + H+ + Cl−

[0042] HOCl reacts with hydrogen peroxide to produce singlet oxygen, since the amino acid histidine is present in the active site of SOD and catalase, singlet oxygen reacts with this amino acid and inactivates these two enzymes at the membrane surface. By inactivating SOD, more singlet oxygen is available for the HOCl reaction and the LPO process is increased.

[0043] HOCl + •O → •OH + O2 + Cl−

[0044] HOCl + Fe2+ → •OH + Fe3+ Cl−

[0045] The extrinsic pathway of apoptosis is also initiated by the activation of the Fas receptor by singlet oxygen, leading to the activation of caspase-7 and caspase-3.

[0046] Enhancement of the immune response: HOCl selectively increases the expression of danger signals in cancer cells, which leads to increased presentation of cancer cell antigens, eliciting and inducing an antitumor response from cytotoxic T cells. Pathophysiological levels of HOCl of 200 μM / L lead to DNA and histone damage. In addition, HOCl exposure results in an immediate and sustained cytosolic accumulation of calcium, which in turn leads to increased production of cytokines and chemokines. HOCl also induces dendritic cell differentiation and macrophage differentiation to the M1 phenotype.

[0047] Calpain activation: Calpains are a group of calciumsensitive cysteine proteases that play important roles in various cellular processes, including apoptosis, autophagy, and inflammation. Hypochlorous acid (HOCl) activates calpains by increasing intracellular calcium. Activated calpains degrade cytoplasmic membranes and nuclear substrates, leading to cellular disruption and ultimately apoptosis. Calpain 1 degrades the Na+ / Ca2+ exchanger NCX in mitochondrial membranes, which cleaves the BH3interacting domain death agonist (Bid), resulting in the release of cytochrome c and apoptosis. Calpain activation and its function in the processing of ApoptosisInducing Factor are also implicated in various cell death pathways.

[0048] In addition, HOCl, through calcium-dependent mechanisms, induces mitochondrial permeability transition, lysosomal rupture, and cell death. In fact, HOCl causes a wide range of oxidative changes and the formation of chlorinated products, leading to changes in the structure of proteins and enzyme activity, thus affecting the performance of biological systems.

[0049] Hypochlorous acid (HOCl) is an effective anticancer agent, but its application in clinical treatments is limited due to its high instability. Given the importance of HOCl in cancer treatment and the importance of its pulmonary delivery, the development of a new drug delivery system to improve the efficiency of HOCl delivery in a stable manner with a continuous and controlled release feature and to reduce side effects is worthy of attention.

[0050] The nanomedicine designed in this invention is based on alginate nanoparticles containing perfluoropentane (PFP) with a folic acid-conjugated chitosan coating, which overcomes the side effects of HOCl in non-target tissues and delivers HOCl more effectively to the cancerous tissue. In the design of this nanomedicine, the hybrid polymer structure based on alginate-chitosan conjugated with folic acid in combination with PFP leads to the controlled release of HOCl, since HOCl is a strong oxidant and can be unstable under certain conditions. Encapsulating its components in polymer nanoparticles leads to the formation of HOCl at the desired location and in addition to maintaining its stable structure, due to the presence of folic acid on the chitosan surface, this structure reaches cancer cells in a targeted manner and after connecting using the explosion caused by ultrasound waves, the components react together and HOCl is formed at a specific concentration at the tumor site.

[0051] Considering the characteristics of hypochlorous acid in cancer treatment and the importance of designing a special drug delivery system for pulmonary drug delivery, the objectives of this invention are as follows:

[0052] 1. Designing a suitable nanodrug for pulmonary and targeted delivery of hypochlorous acid to the patient's lung

[0053] 2. Sustained production of hypochlorous acid at the cancer site

[0054] 3. Control and regulation of drug release

[0055] 4. Increasing the efficiency of drug deliverySolution of Problem

[0056] - Alginate-Chitosan Nanoparticle Fabrication

[0057] Alginate-chitosan structure containing Ca(OCl)2 and salicylic acid is fabricated in a two-step optimized method based on Nano-emulsion method and coating using Ionic gelation technique.

[0058] To fabricate PFP-containing alginate particles, first 1 ml volume of 5% calcium chloride solution containing 0.1% polyvinyl alcohol, different concentrations of Ca(OCl)2 solution and 1-5% Perfluoro Pentane are mixed together using probe sonicator and after complete mixing, it is nebulized by peristaltic pump with sterile components into sodium alginate solution with concentration 1.23% (w / w) and trehalose with concentration 8.25% with pH = 5 using jet nebulizer.

[0059] To prepare folic acid conjugated chitosan, first 44 mg of folate is well dissolved in 15 ml of anhydrous DMSO, and then 10 mM EDC (Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) is slowly added to the solution and stirred well for one hour at room temperature. Then, 5 mM chitosan sodium acetate 5% v / v at pH = 5 is added to the solution and stirred at 30 °C for 16 hours. This allows the carboxylic groups in folate to react with free amine groups in chitosan to form an amide bond, thereby conjugating folate to chitosan. The reaction mixture is adjusted to pH = 9.0 by dropwise addition of diluted sodium hydroxide and dialyzed against a solution containing sodium phosphate buffer with pH = 7.4 for 3 days using a dialysis bag (molecular weight cut-off, 12 kDa). The resulting solution is stored at -48°C for 12 hours. It is then dried using a freeze dryer. Then, the folic acid-conjugated chitosan is dissolved in a PBS solution containing 2.5 mM salicylic acid to load the acidic part of the formulation into the chitosan.

[0060] Ionic gelation technique was used to coat alginate with acid-containing chitosan, whereby 4 ml of salicylic acid-containing chitosan solution was added dropwise to the alginate solution (which was being stirred at 500 rpm). The particle suspension was centrifuged at 20,000 rpm for 30 min to increase stability, ensure adequate drug loading, and optimize drug release, and then washed three times. The washing and centrifugation process was repeated two more times to ensure removal of all residual impurities and excess reagents that may be present in the suspension.

[0061] - Measurement of folate conjugated to chitosan

[0062] The folate content in the folate conjugated chitosan formulation was determined using a fluorometric method with a multi-mode microplate reader (Molecular Devices, USA). 2 mg of folate conjugated chitosan was dissolved in 10 ml of a mixture of dimethyl sulfoxide (DMSO) and dichloromethane (DCM) in a ratio of (4:1) for 6 hours using a vortex. The samples were centrifuged and the supernatant was filtered and its absorbance was measured. A folic acid calibration curve was prepared at λ max = 364 nm and the total folic acid content in folate conjugated HOCL-PFP-Alginate-Chitosan nanoparticles was determined.

[0063] The percentage of folic acid content was calculated using the following formula.

[0064] Conjugated folate amount (%) = Measured folate amount in the nanoparticle ÷ Total folate content used in the nanoparticle preparation.

[0065] The amount of folic acid conjugated to chitosan was calculated to be 78.5 ± 0.09%.

[0066] - Investigation of the properties of HOCL-PFP-Alginate-Chitosan nanoparticles:

[0067] The particle size distribution and zeta potential of the nanoparticles were analyzed by DLS method to measure the surface charge using a Malvern zeta Nano ZS (Zeta-sizer 3000HS, Malvern Instruments, Malvern, UK). DLS was performed based on quasi-elastic light scattering (QELS) at 25°C and a scattering angle of 90°. All samples were measured at 25°C in triplicate. The average hydrodynamic size of the nanoparticles was measured to be 1092 ± 170.3 nm.

[0068] The optimal particle size is shown to be 1092 ± 170.3 nm [Pic. 2].

[0069] The physicochemical parameters of HOCL-PFP-Alginate-Chitosan nanoparticles are shown in [Table 1].

[0070] The HOCL-PFP-Alginate-chitosan-NPs nanoparticles had an average size of 954 nm [Table 1]. The relatively low poly dispersity index (PDI) of each nanoformulation (<0.3) indicates that the nanoparticles have a narrow size distribution. High-performance liquid chromatography (HPLC) was used to evaluate the encapsulation efficiency (EE%) of the HOCL-PFP-Alginate-chitosan-folate-NPs nanoparticles. The encapsulation efficiency was 98.38%.

[0071] - Morphological study of HOCl-PFP-Alginate-Chitosan nanoparticles

[0072] Transmission electron microscopy was used to examine the morphology of the obtained nanoparticles. TEM imaging (TEM, H-7650; Hitachi, Tokyo, Japan) was used to investigate the size, shape, and morphology of HOCL-PFP-Alginate-chitosan-NPs. For this purpose, samples were prepared by drop-casting method without using dye. Before imaging, nanoparticles were placed on a carbon-coated copper grid (400 Mesh) and dried at room temperature.

[0073] SEM imaging was used to confirm the formation of HOCL-PFP-Alginate-Chitosan NPs nanoparticles and visualize their size, shape, and surface morphology. SEM was obtained using a scanning electron microscope (SEM) (Hitachi S4700, Hitachi Scientific Ltd, Tokyo, Japan) at 20 kV and magnification of 12000. For SEM analysis, HOCL-PFP-Alginate-Chitosan nanoparticles were diluted with ultra-pure water. One drop of each sample was placed directly on the aluminum slide of a Quanta 200 ESEM (FEI, USA) SEM and dried at 24–40 °C. During imaging, the air pressure was maintained at 1.3–13.0 mPa.

[0074] TEM analysis results showed that HOCL-PFP-Alginate-chitosan-NPs nanoparticles have a core-shell structure with a dark core and a light shell around it and a relatively uniform particle size (approximately 950 to 1100 nm). SEM imaging results showed that the nanoparticles were uniformly dispersed and had a smooth surface without holes or cracks. On the other hand, the DLS results were consistent with the SEM and TEM results.

[0075] - Measurement of the concentration of HOCl released from the nanoparticles

[0076] The concentration of HOCl and OCl- was measured spectrophotometrically and the concentrations were determined using the Beer-Lambert law. In order to prepare the samples, a 5 ml sample of each concentration of HOCl-PFP-Alginate-Chitosan nanoparticles (200, 400, 800, and 1000 mg) was taken and its volume was brought to 100 ml with autoclaved deionized water. The nanoparticle structure sample without HOCl was considered as a blank sample. Each sample was divided into four 10 ml samples to check the reproducibility of the experiment and each experiment was repeated 4 times. Each sample was subjected to sonication and then its absorbance was measured.

[0077] HOCl concentration at 234 nm and OCl- concentration at 292 nm were measured. For this purpose, a quartz cuvette was used and the path length was considered to be 1 cm. The results were reported in mg / L.

[0078] The results of determining the concentration of HOCl and OCl- are shown in [Table 2].

[0079] - HOCl release profile investigation using ultrasound waves

[0080] HOCl release profile was determined using dialysis method. For this purpose, 1 ml of nanoparticles was sealed in a dialysis bag (MWCO 3000 Da) and immersed in 400 ml of buffer solution (pH = 6.5) at 37 °C with continuous shaking in a water bath, and the dialysis bag was placed in the water bath under ultrasound waves of 1.1 MHz 55.8 mW / cm for 10 minutes.

[0081] At time intervals of 0, 50, 100, 150, 200, 250, 300 and 350 minutes, 1 ml of the medium was collected and replaced with 1 ml of fresh buffer.

[0082] The release profiles of Ca(OCl)2 from the nanoparticle using ultrasound waves and the formation of HOCl show that the nanoparticle disintegrates upon exposure to ultrasound waves and releases Ca(OCl)2, which forms HOCl upon reaction with acid [Pic. 3]. The cumulative release rate of HOCl after irradiation of the nanoparticle with ultrasound waves increases rapidly with increasing Ca(OCl)2 concentration.

[0083] - Cytotoxicity Study

[0084] Cytotoxicity study was performed using the MTT method. For this purpose, A549 cells were seeded in 96-well plates at a density of 104 × 1 cells per well and incubated for 12 hours.

[0085] Cytotoxicity study of A549 cells was performed after treatment with PFP-Alginate-Chitosan NPs, HOCl, and HOCl-PFP-Alginate-Chitosan NPs in the presence of ultrasound waves at different concentrations for 24, 48, and 72 hours [Pic. 4]. Each experiment was repeated three times. Ultrasound waves were applied 1 hour after different treatments at a power of 3 watts for 15 minutes. After incubation of the cells for 24, 48 and 72 hours, 20 μl of MTT solution ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) at a concentration of 5 mg / ml was added to each well and incubated for 4 hours. After the culture medium was replaced with 150 μl of dimethyl sulfoxide (DMSO), the optical density of the cells (OD) was measured using a Synergy HTX Nanodrop device (Bio Tek, USA) at a wavelength of 490 nm.

[0086] IC50 at 24, 48 and 72 hours was calculated in different groups by GraphPad Prism software.

[0087] PFP-Alginate-Chitosan NPs were not cytotoxic at any concentration, indicating that PFP-Alginate-Chitosan NPs are suitable for in-vivo applications [Pic. 4]. The cytotoxicity of HOCl-PFP-Alginate-Chitosan NPs significantly increased with increasing nanoparticle concentration and time, leading to a decrease in cell viability [Pic. 4] (a) and showed high cytotoxicity in A549 cells. Based on the results of the MTT assay, free HOCl showed higher cytotoxicity than HOCl-PFP-Alginate-Chitosan nanoparticles at the same concentration of HOCl [Pic. 4] (b). This indicates that HOCl contained in the NPs is gradually released into the surrounding environment, which explains the relative increase in cytotoxicity over time. HOCl-PFP-Alginate-Chitosan nanoparticles also showed higher cytotoxicity after ultrasound irradiation (1.1 MHz; 55.8 mW / cm2; 10 min) at 48, 24, and 72 h [Pic. 4] (a)..

[0088] Notably, no significant cytotoxicity was observed in A549 cells after ultrasound irradiation, indicating that the ultrasound irradiation dose had no obvious toxicity and was safe for the cells.

[0089] PBS solution and drug-free nanoparticles (PFP-Alginate-Chitosan) were used as controls to examine cell viability in various concentrations of HOCl have been used in the range of 200 to 1000 ppm.

[0090] - Examination of the expression of proteins responsible for apoptosis in A549 cells

[0091] Western blot test was used to examine the effect of nanoparticles on apoptosis. For this purpose, A549 cells were treated with HOCl-PFP-Alginate-Chitosan NPs for 48 and 24 hours. The cells were lysed using RIPA at 4°C. The BCA protein assay kit was used to measure protein concentration. SDS-PAGE was used to separate proteins. After electrophoresis, the proteins were transferred to PVDF membranes by electroblotting and then blocked in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.2% Tween-20, Sigma) containing 5% nonfat milk. In order to perform Western blotting, PVDF membranes were used. PVDF membranes were incubated with primary antibodies against Caspase-3, PARP-1, Bax, BCL2 and GAPDH (1:1000) at 4°C overnight and then washed twice with TBST. Then, the membranes were incubated with peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour and after washing, ECL reagent was used to detect protein bands.

[0092] Bcl-2 family and caspase-3 are considered as a major hallmark of apoptosis. In order to investigate the effect of HOCl-PFP-Alginate-Chitosan NPs on Bcl-2 family protein expression and caspase-3 activation in A549 cells, these cells were exposed to 1000 ppm of HOCl-PFP-Alginate-Chitosan nanoparticles for 48 hours.

[0093] The expression levels of apoptotic proteins, including the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2, were measured. The results showed that HOCl-PFP-Alginate-Chitosan NPs increased the expression of caspase-3, PARP-1, and Bax, but decreased the expression of Bcl-2 in A549 cells [Pic. 5].

[0094] - Investigation of the penetration and effect of HOCl-PFP-Alginate-Chitosan NPs on orthotopic murine model of lung cancer

[0095] To establish an orthotopic murine model of lung cancer, A549 cells were transfected with luciferase and injected intrathecally into mice. Confirmation of the initial deposition of cancer cells in the lungs, including confirmation of bioluminescence of transfected cancer cells in live and anesthetized animals, was performed using the LV200 LUMINOVIEW imaging system. Bioluminescence technique was used to confirm the formation of cancer cells in the lungs and other organs. For this purpose, after killing the mice, the lungs and other organs of the mice were removed and examined. The findings showed that tumors were formed in the lungs of the mice and approximately 5% of the mice had brain metastases. The size of the lung tumors was quantitatively analyzed.

[0096] Histological examination of lung tissues showed that untreated tumor-bearing mice showed the presence of numerous tumor nodules that compressed the adjacent bronchi and alveoli. These tumors consisted of pleomorphic cells with abundant eosinophils and large irregular nuclei. In addition, they contained several prominent nuclei. The tumor cell masses were interspersed with a fine connective tissue stroma and small capillary networks. In contrast, the lungs of healthy (control) mice showed well-defined features with preserved epithelium. The alveoli were open and free of any signs of inflammation.

[0097] The distribution of inhaled HOCl-PFP-Alginate-Chitosan NPs was investigated using a Collison (BGI) nebulizer (CH Technologies). HOCl-PFP-Alginate-Chitosan nanoparticles were nebulized into the lung at a flow rate of 2 L / min. Ultrasound stimulation was performed one hour after drug nebulization. (The ultrasound device parameters were as follows: 1 cm2 focal area, pulsed wave mode, 50% duty cycle, 1.1 MHz, 55.8 mW / cm2, and 2.5 cm focal length). The distribution of nanoparticles in different organs was investigated up to 10 days after drug nebulization.

[0098] The findings show that administration of HOCl-PFP-Alginate-Chitosan NPs by inhalation significantly increased the accumulation of these nanoparticles in the lungs and reduced their accumulation in other organs. Also, analysis of the content of HOCl-PFP-Alginate-Chitosan NPs per gram of tissue in different organs showed their preferential accumulation in the lungs after inhalation administration.

[0099] During treatment, there was a significant rapid increase in tumor volume for both the control and PFP-Alginate-Chitosan NPs groups. However, administration of free HOCl or HOCl-PFP-Alginate-Chitosan NPs showed significant inhibition of tumor growth, the effect of which was significantly enhanced after ultrasound irradiation [Pic. 6] (a).

[0100] No significant difference was observed between the control groups under ultrasound and without ultrasound irradiation, indicating that the administered ultrasound irradiation dose was safe.

[0101] On day 10, the tumor weight in the groups treated with free HOCl or HOCl-PFP-Alginate-Chitosan NPs was 29.12% and 2.32% of the tumor weight of the control group, respectively [Pic. 6] (c).

[0102] The intratumoral accumulation and distribution of the drug were significantly increased by ultrasound irradiation, leading to inhibition of tumor proliferation. It is worth mentioning that the in vivo findings indicate that HOCl-PFP-Alginate-Chitosan nanoparticles have higher therapeutic efficacy compared to free HOCl.

[0103] The survival rate of mice was measured during the experiment, [Pic. 6] (b) shows the survival rate of mice in different treatment groups. Notably, treatment with HOCl-PFP-Alginate-Chitosan nanoparticles under ultrasound irradiation resulted in a significant increase in survival up to 40 days.

[0104] TUNEL staining was performed using an apoptosis detection kit to evaluate the histological characteristics of lung tumor tissues after treatment. The results of TUNEL staining showed increased apoptosis in tumors of mice treated with HOCl-PFP-Alginate-Chitosan NPs and ultrasound irradiation. The findings of this study indicate that the combination of HOCl-PFP-Alginate-Chitosan NPs and ultrasound irradiation effectively penetrates into extravascular tumor tissues through the tumor vascular endothelial gap and directly targets tumor tissue through specific binding to the folate receptor, thereby leading to effective tumor treatment.Advantage Effects of the Invention

[0105] - Targeted pulmonary delivery of hypochlorous acid to the patient's lung

[0106] - Sustained production of hypochlorous acid at the site of cancer

[0107] - Possibility of inhaled administration of nanomedicine

[0108] - Significant reduction in drug side effects

[0109] - Rapid action and destruction of lung cancer cells

[0110] - Precise spatio-temporal control of drug release

[0111] - Control of drug delivery cycle

[0112] - Increased therapeutic efficacy through pulmonary drug delivery

[0113] - Drug release and increased tumor penetration efficiency through mechanical effect caused by ultrasonic wave radiation

[0114] Shows a general flowchart of the proposed method.

[0115] Declares a schematic diagram of the nanoparticle production process.

[0116] Shows a general flowchart of the claimed solution and its various stages of developing the claimed nanodrug.

[0117] Displays a schematic diagram of the steps for manufacturing HOCl-PFP-Alginate-Chitosan nanoparticles.Examples

[0118] The invented nanomedicine can be used to start clinical trials in humans. For this purpose, first the tumor sites are identified using pETscan, then HOCl-PFP-Alginate-Chitosan NPs nanoparticles with a specific concentration are inhaled into the patient's lungs and locally, using ultrasound waves with a specific frequency, the drug is released at the tumor site, leading to the induction of apoptosis and the destruction of cancer cells.

[0119] [Pic. 1] Here the mechanism of the drug after administration is captured.

[0120] [Pic. 1]

[0121]

[0122] [Pic. 2] Displays a graph of the average hydrodynamic size of HOCl-PFP-Alginate-Chitosan nanoparticles by (DLS) method.

[0123] [Pic. 2]

[0124]

[0125] [Pic. 3] Depicts a graph of HOCl release profile from HOCl-PFP-Alginate-Chitosan nanoparticles using ultrasound waves at 350 min.

[0126] [Pic. 3]

[0127]

[0128] [Pic. 4] Shows cell viability graph of A594 cells treated with HOCl-PFP-Alginate-Chitosan NPs for 24, 48 and 72 h. (a) Cell viability graph of A549 cells treated with HOCl for 24, 48 and 72 h. Data are presented as mean ± SD (n = 3).

[0129] [Pic. 4]

[0130]

[0131] [Pic. 5] Captures the western blot analysis of Caspase 3, PARP-1, Bax, Bcl2 at 24 and 48 h after treatment of A594 cells with HOCl-PFP-Alginate-Chitosan NPs. Data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001

[0132] [Pic. 5]

[0133]

[0134] [Pic. 6] Shows the analysis of relative tumor volume in tumor-bearing mice that were treated (n=3) (a). Analysis of survival of mice under different treatments (n=5) (b). Weight of tumors isolated from tumor-bearing mice on day 10 (n=5) (c).

[0135] [Pic. 6]

[0136]

[0137] More data regarding the process and the components is gathered in the tables listed below:

[0138] [Table 1] Particle size distribution; surface charge, encapsulation efficiency with Ca(OCl)2 and Salicylic acid.

[0139]

[0140] [Table 2] Determination of HOCl and OCl concentrations by spectrophotometry.

[0141]

[0142] [Table 3] IC50 of A549 cells treated with free HOCl and HOCl-PFP-Alginate-Chitosan NPs for 24, 48 and 72 h. Results are presented as mean ± standard deviation.

[0143]

[0144] HOCl-PFP-Alginate-Chitosan NPs, a bilayer structure, consists of PFP-containing alginate in the inner layer and folate-conjugated chitosan in the outer layer, with calcium hypochlorite Ca(OCl)2 in the alginate core of the nanoparticle and salicylic acid in the chitosan coating. These nanoparticles are fabricated in a two-step process based on Nano-emulsion and coating using Ionic gelation technique and are used in the treatment of lung cancer.

[0145] Pulmonary delivery of HOCl-PFP-Alginate-Chitosan NPs with the help of ultrasound waves has led to increased efficiency of HOCl delivery to the tumor site, which leads to the treatment of lung cancer. This drug can be used in medical facilities, clinics, hospitals and lung cancer research centers. Healthcare and pharmaceutical companies who prioritize supporting newly developed cancer treatments can partake in the production process of this nanodrug.

Claims

An active nanoparticle drug delivery solution is designed for targeted lung cancer treatment which provides controlled release of HOCl at tumor site.According to claim 1, the designed nanodrug product is obtained through the fabrication of alginate chitosan nanoparticles via nano-emulsion and ionic gelatin procedure.According to claim 2, after the fabrication is complete, the folate conjugated to chitosan are measured via Fluorometry to determine the amount of the targeting agent present in the nanoparticles.According to claim 3, the process then involves an investigation of properties of the produced HOCl-PFP alginate chitosan nanoparticles.According to claim 4, next phase of the process includes the morphological study of HOCl-PFP alginate chitosan nanoparticles to understand their structure and stability.According to claim 5, after the study of the produced nanoparticles, the concentration of HOCl released from the nanoparticles is measured.According to claim 6, a crucial part of the process is the HOCl release profile investigation using dialysis method via ultrasound waves.According to claim 7, next a cytotoxicity assessment is performed via the MTT method to provide insights into the efficacy and safety of the nanodrug.According to claim 8, then a western blot is performed for evaluating expression of proteins responsibe for apoptosis in A549 cells.According to claim 9, the last step is the investigation of penetration and effect of HOCl-PFP-Alginate-Chitosan NPs on orthotopic murine model of lung cancer to provide information on their in vivo behavior and clinical application.

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