Temperature- and ph-sensitive, BPA-targeted, chitosan–poly(n-isopropylacrylamide)–FPBA core-shell polymeric nanoparticles capable of forming COF structures for use in the BNCT therapeutic method.

Abstract

This invention, entitled "Temperature- and pH-Sensitive BPA-Targeted Chitosan–Poly(N-isopropylacrylamide)–FPBA Core-Shell Polymeric Nanoparticles Capable of Forming COF Structures in the BNCT Therapeutic Method," pertains to an anti-cancer pharmaceutical composition utilizing novel drug delivery methods. The application of the novel BNCT method is advantageous for cancers such as glioblastoma due to the challenge of crossing the blood-brain barrier. BNCT is a dual and targeted method wherein cancer cells, following the accumulation of ¹⁰B, are irradiated with thermal neutrons. Loading boron-containing compounds into nanoparticles can deliver a high concentration of boron to human glioblastoma cells. Temperature- and pH-sensitive nanoparticles of succinylated chitosan–poly(N-isopropylacrylamide) targeted with BPA are our concept for achieving endocytosis via sialic acid receptors on the surface of glial cells and for the targeted delivery of boron to these cells. By designing temperature- and pH-sensitive, BPA-targeted Chitosan–Poly(N-isopropylacrylamide)–FPBA core-shell polymeric nanoparticles with the capability of forming COF structures in order to simultaneously deliver BPA and FPBA, we aim to utilize the polymeric boron content to perform treatment via the BNCT method. Consequently, damage to healthy cells is reduced to a minimum, and even to zero.

Description

Temperature- and pH-Sensitive, BPA-Targeted, Chitosan–Poly(N-isopropylacrylamide)–FPBA Core-Shell Polymeric Nanoparticles Capable of Forming COF Structures for Use in the BNCT Therapeutic Method.

[0001] This composition falls within Section A, namely Human Necessities, and the subsection of health and hygiene, and pertains to an anti-cancer pharmaceutical composition utilizing novel drug delivery methods. The international classification of this invention, under the International Patent Classification (IPC) system on the website of the World Intellectual Property Organization (WIPO), is A61K41 / 0095.

[0002] Malignant gliomas, particularly glioblastoma multiforme (GBM), still exhibit resistance to conventional treatments, including surgery, chemotherapy, radiotherapy, immunotherapy, and gene therapy. Conventional surgical methods are limited to the main tumor mass; however, they do not eliminate the microscopic malignant cells that have invaded adjacent healthy tissues, leading to the recurrence of the disease and tumor. Chemotherapy and radiotherapy methods are also incapable of eliminating invasive tumor cells. Since they require a high radiation dose, they result in severe brain damage. Despite these aggressive treatments, only a few patients with glioblastoma multiforme survive for less than one year. Glioma cells invade the brain's extracellular environment due to its biochemical characteristics, eventually encompassing the entire brain tissue. To treat glioblastoma, molecular methods have been developed that selectively target malignant tumor cells in a manner that has minimal effect on adjacent cells and tissues. Brain tumors cause disruption in the function of the blood-brain barrier. Researchers have shown that nanoparticles are capable of crossing the blood-brain barrier and can be a suitable method for drug delivery and the treatment of brain tumors. For this reason, the use of nanoparticles for brain cancer treatment has garnered significant attention today.

[0003] A major obstacle to the success of brain tumor treatment is the presence of the blood-brain barrier. The blood-brain barrier is highly permeable to water, carbon dioxide, oxygen, and lipid-soluble substances like alcohol. The blood-brain barrier has low permeability to electrolytes but is completely impermeable to plasma proteins and many non-lipid-soluble large organic molecules. This selective permeability of the blood-brain barrier results from the tight junctions formed within the capillary endothelium in the brain. The blood-brain barrier exists in the capillary membrane of all brain regions except for certain areas of the hypothalamus and the pineal gland. However, it is important to note that the presence of a selectively disrupted blood-brain barrier presents an opportune moment for delivering the best therapeutic agents. Just as many therapies and nanoparticles emphasize the use of enhanced permeability and the retention effect for the accumulation of particles and pharmaceutical agents over time, owing to the ineffective vasculature and lymphatic system of cancer in other parts of the body, a similar disruption can be utilized for drug delivery to brain tumors. The use of a nanoparticle system in drug delivery offers significant benefits. Nanocarriers can penetrate cellular and tissue pores through the smallest capillary vessels without any difficulty to reach target tissues such as the brain, spinal cord, lungs, spleen, lymph, and liver. In order to avoid rapid clearance of the nanoparticles by phagocytes, their circulation time in the blood is also prolonged. Chemotherapy and radiotherapy methods are also incapable of eliminating invasive tumor cells. To treat glioblastoma, molecular methods were developed that selectively target malignant tumor cells in such a way as to have the least effect on adjacent cells and tissues.

[0004] Boron Neutron Capture Therapy (BNCT) is a suitable therapeutic method for selectively eliminating tumor cells. BNCT has been used in the treatment of gliomas and brain metastases, melanoma, and recently in the treatment of head and neck, liver, lung, and thyroid cancers. BNCT is a selective and lethal therapeutic method for tumor cells using thermal neutron beams, and the efficacy of this method depends on the cellular uptake amount of ¹⁰B. BNCT has two components for treatment: the first component is the use of the naturally stable boron, ¹⁰B; the second component is a low-energy neutron beam.

[0005] The design of boron-containing polymeric systems is the most suitable method for the targeted delivery of boron to cancer cells. By designing core-shell polymeric nanoparticles of chitosan–poly(N-isopropylacrylamide)–FPBA, targeted with BPA, which are sensitive to temperature and pH and have the capability to form COF structures for the simultaneous delivery of BPA and FPBA, we aim to utilize the polymeric boron content to perform treatment via the BNCT method.

[0006] BNCT is a selective and lethal therapeutic method for tumor cells using thermal neutron beams, and the efficacy of this method depends on the cellular uptake amount of ¹⁰B. When ¹⁰B is irradiated with low-energy thermal neutrons, a destructive, high-energy alpha particle and a lithium atom nucleus are released within the tumor cell due to the fission of the boron nucleus. The energy from the α-particles (⁴He) and ⁷Li ions produced during neutron capture causes the death of cancer cells by destroying their cellular DNA molecules. The alpha particle has a Linear Energy Transfer (LET) of 150 keV, and the lithium ion has an LET of 175 keV. These particles cover a short range (5-9 micrometers) in tissue, consequently limiting the energy penetration to the diameter of a single cell. Therefore, theoretically, it is possible to act selectively and destroy tumor cells without affecting healthy cells, the mechanism of which is illustrated in Figure (1-1). This therapeutic method requires two crucial and fundamental parameters: having a suitable neutron source and achieving a ¹⁰B-carrying drug. The boron-containing compound in the tumor cell, following thermal neutron irradiation, leads to the destruction of tumor cells without damaging adjacent healthy cells. When ¹⁰B is present at the cellular surfaces or in the interstitial space of the tumor tissue, its effect on the cell is reduced compared to when it accumulates inside the tumor cell. Therefore, the successful treatment of cancer by BNCT requires the selective delivery of relatively large quantities of ¹⁰B compounds to malignant tumor cells. The required output concentration for effective treatment is in the range of 20-30 µg / g. At the same time, the boron concentration in the surrounding healthy tissue must be low to minimize damage.

[0007] To date, chitosan nanoparticles have not been used as a drug delivery system in BNCT that is intelligently sensitive to temperature and pH. The only case that bears some similarities to this invention is an article titled "Boron phenylalanine targeted chitosan–PNIPAAm core–shell thermo-responsive nanoparticles: boosting drug delivery to glioblastoma in BNCT," which was published on February 14, 2022 (1 Bahman 1400), in the journal Drug Development and Industrial Pharmacy by myself and my colleagues (respectively: Soleimanbeigi, Doosti, Hassanzadeh, Mirian, Varshosaz, Kasesaz, Rostami). I will elaborate further on the points of differentiation between that article and the current pharmaceutical composition. Due to the new boron-containing compound used in the chitosan backbone and in the shell of this drug delivery system, in addition to increasing the concentration of boron atoms delivered to the target cell, this compound forms an imine bond with the chitosan backbone. Through this, the pH-sensitive feature is created in our intended drug delivery system. This matter contributes to a more complex and precise drug delivery process and further helps prevent premature drug release at incorrect locations. According to the 2021 article titled "Chitosan-Based Biocompatible Copolymers for Thermoresponsive Drug Delivery Systems: On the Development of a Standardization System" by Lorenzo Marsili et al., the difficulty in predicting the temperature-dependent behavior and the thermal transition points, 'TCP' and 'LCST', has hindered the clinical application of such drugs from demonstrating the required efficacy. Therefore, by incorporating pH sensitivity alongside temperature sensitivity, the weakness of such a system can be compensated for. Furthermore, with the modifications made, the boronated compound (FPBA) used in the new formula has the capability to be converted into COF structures under controlled reactions, which results in a significantly lower molecular weight and the capacity for loading higher concentrations of boronated and anti-cancer drugs. The intelligent placement of the components of the new pharmaceutical composition in one formulation and their synergistic effect on one another has led to a remarkable increase in the efficacy of the drug BPA and the boron-containing compound FPBA in recent laboratory results, in such a way that it has made definitive treatment possible in laboratory samples where it had a low percentage with previous compounds.

[0008] This invention, entitled "Temperature- and pH-Sensitive BPA-Targeted Chitosan–Poly(N-isopropylacrylamide)–FPBA Core-Shell Polymeric Nanoparticles Capable of Forming COF Structures in the BNCT Therapeutic Method," pertains to an anti-cancer pharmaceutical composition utilizing novel drug delivery methods. The application of the novel BNCT method is advantageous for cancers such as glioblastoma due to the challenge of crossing the blood-brain barrier. BNCT is a dual and targeted method wherein cancer cells, following the accumulation of ¹⁰B, are irradiated with thermal neutrons. Loading boron-containing compounds into nanoparticles can deliver a high concentration of boron to human glial cells. Temperature- and pH-sensitive nanoparticles of succinylated chitosan–poly(N-isopropylacrylamide) targeted with BPA are our concept for achieving endocytosis via sialic acid receptors on the surface of glial cells and for the targeted delivery of boron to these cells. By designing temperature- and pH-sensitive, BPA-targeted Chitosan–Poly(N-isopropylacrylamide)–FPBA core-shell polymeric nanoparticles with the capability of forming COF structures in order to simultaneously deliver BPA and FPBA, we aim to utilize the polymeric boron content to perform treatment via the BNCT method. Consequently, damage to healthy cells is reduced to a minimum, and even to zero.

[0009] The principal drawbacks of conventional cancer treatment methods include the high toxicity of anti-cancer drugs, owing to their systemic distribution and indiscriminate release towards both healthy and cancerous cells. The application of the novel therapeutic method, Boron Neutron Capture Therapy (BNCT), is advantageous for cancers such as glioblastoma, in view of the inability to cross the blood-brain barrier or the undesirable lesions that remain following conventional therapeutic methods. Furthermore, due to their low solubility in water and the use of organic solvents, anti-cancer drugs are accompanied by undesirable side effects such as venous irritation and respiratory distress. Therefore, the design of nano-based drug delivery systems for successful cancer treatment becomes a necessity. In order to be used in targeted drug delivery systems, nanoparticles must be designed and engineered in a way that enables them to cross blood vessels, transport the drug into the cell, and also possess the capability for correct drug release. Additionally, these nanoparticles must have stability and a long lifespan in the blood and possess the ability to resist tissue macrophages. Another property that receives significant attention in nanoparticles is their non-toxic, biodegradable, and biocompatible nature, as well as their non-invasive character towards healthy body cells. These characteristics enable nanoparticles to aptly perform targeted drug delivery, and on the other hand, reduce the side effects of using these carriers to a minimum.

[0010] In stimuli-responsive systems, the existing stimulus can be external or internal. Among the systems responsive to external stimuli, one can name temperature-sensitive systems, magnet-sensitive systems, ultrasound-sensitive systems,

[0011] light-sensitive systems, and electric field-sensitive systems. Internal stimuli include changes in pH, changes in electrical potential, and enzymatic changes in cancer cells, based on which pH-sensitive, Redox-sensitive, and Enzyme-sensitive systems have been respectively developed, which respond to stimuli present in the target tissue, such as temperature, pH, enzymes present in the tissue, etc., and cause drug release. Stimuli-responsive systems that are capable of changing their structure and consequently their properties in response to physiological variables attract greater attention as chemical sensors and therapeutic systems.

[0012] In this design, boron phenylalanine is used as a targeting agent. Considering the extensive and effective application of chitosan as a drug delivery system, we decided to increase the polymeric boron content for performing treatment via the BNCT method by synthesizing a temperature- and pH-sensitive BPA-targeted Chitosan-Poly(N-isopropylacrylamide)-FPBA conjugate. It should be noted that, to date, dual temperature- and pH-sensitive systems based on chitosan, which use two boron-containing compounds—boron phenylalanine as a targeting agent in addition to the main drug, and FPBA as a pH-sensitive agent (due to the formation of an imine bond between the carboxyl group of the aldehydic agent and the NH₂ group present in the chitosan backbone)—have not been used in the BNCT therapeutic method.

[0013] Given the higher concentration of conjugated FPBA in the chitosan backbone and the smaller size of this boron-containing compound compared to BPA, this compound, under controlled reactions, has the ability to form COF structures with a significantly higher loading capacity for anti-cancer drugs, in addition to boronated drugs, following boroxine or boronate ester bonds. The growth of these structures occurs via the B(OH)₂ functional groups in the FPBA compound, in such a way that the BPA-targeted Chitosan-Poly(N-isopropylacrylamide) conjugate acts as a coating on this COF structure. Since boroxine or boronate ester bonds are stable at physiological pH (7.4) and have hydrolytic properties at acidic pH (≤5), these structures have the capability to form a hydrogel. It should be noted that, following a synergistic effect, the FPBA compound loaded in the shell of the drug delivery system is released inside cancer cells due to the acidic pH, and the release of the BPA drug loaded in the core of this system occurs following the temperature increase of the cancer cells. This simultaneous delivery of two boron-containing compounds leads to toxic effects on the targeted tumor cells upon irradiation with thermal neutrons. In addition to the external stimuli of increased temperature and the acidic pH of the cancer cell tissue, BPA, as a targeting agent, aids in the targeted delivery of the boronated drug to tumor cells on whose surface sialic acid receptors are overexpressed. On the other hand, the content of the boron-containing compound within the drug delivery system is increased. Consequently, damage to healthy cells is reduced to a minimum, and it can even be said that it is reduced to zero.

[0014] Temperature- and pH-sensitive Chitosan–Poly(N-isopropylacrylamide)–FPBA nanoparticles targeted with BPA were synthesized for the simultaneous delivery of BPA, FPBA, and methotrexate drugs to the U87MG cell line (human glioma). Two temperature- and pH-sensitive nanoparticle systems were prepared with different ratios of chitosan and poly(N-isopropylacrylamide). The drug-carrying nanoparticles were spherical in shape, with a particle size between 100-200 nm, a low polydispersity index, and the highest loading capacity. The temperature and pH sensitivity of these nanoparticles was clearly observable in the increasing trend of drug release. The cytotoxicity of the methotrexate-carrying nanoparticles (as a model drug) was evaluated on two cell lines, U87MG and A-431. Between the two systems, the CSSU-PNI2 system with a higher polymer ratio demonstrated better temperature-sensitive behavior in all investigations. Compared to non-targeted nanoparticles and the free drug, the boron phenylalanine-targeted nanoparticles had a therapeutic dose of less than 40 µg / mL in vitro on the U87MG cell line. They also showed significantly greater cytotoxic effects on U87MG cells compared to A-431 cells, which confirmed the efficacy of the targeting strategy and the cellular entry of the nanoparticles via endocytosis. The IC50 of the boron phenylalanine-targeted nanoparticles on the U87MG cell line was significantly (P<0.05) lower than that of the non-targeted nanoparticles. In the flowcytometry method, the targeted nanoparticles were taken up considerably more by U87MG cells compared to the non-targeted nanoparticles. Following the hemolysis or hemoglobin destruction test, the hemolysis percentage of the nanoparticles was much less than 5% and within the acceptable range. In view of the obtained results, it can be asserted that the prepared nanoparticles have no interaction with blood cells and do not cause toxicity in the bloodstream.

[0015] The synthesis pathway and the FT-IR and ¹HNMR spectra of the boronated, temperature- and pH-sensitive systems are provided in Figures 1,A to 2,E, respectively.

[0016] In this section, the synthesis pathway of BPA-BOC is provided. As can be seen, this synthesis pathway begins with the boron phenylalanine compound. In this part, the NH₂ group of boron phenylalanine is protected by the carboxyl group of BOC₂O so that in the step of targeting chitosan, it is conjugated to the free NH₂ group of the chitosan backbone via the carboxyl group of BPA.

[0017] In this section, the synthesis pathway of poly (N-isopropylacrylamide) is provided. As can be seen, the polymerization is carried out via the radical reaction of the N-isopropylacrylamide monomer in the presence of AIBN as the initiator for this reaction. In this research project, by controlling the polymerization conditions, we achieved an excellent yield and a suitable molecular weight for the synthesis of poly (N-isopropylacrylamide).

[0018]

[0019] Chart B: The FT-IR spectrum of compound A is provided in this section. According to the FT-IR spectrum, the characteristic band in the 3436 cm⁻¹ region is due to the asymmetric N-H stretching. The presence of C-H is confirmed by the absorption band in the 3028 cm⁻¹ region. The strong band in the 2913 cm⁻¹ region is due to the asymmetric stretching of the CH₂ group. The absorption band in the 1956 cm⁻¹ region indicates the C=C group stretching. The C=O stretching vibration in the carbamate group and the acid group, at 1684 cm⁻¹ and 1720 cm⁻¹ respectively, confirms the N-BOC protected group. The absorption band in the 1333 cm⁻¹ region is due to the bending of the OH group. The absorption bands in the 1161 cm⁻¹, 1130 cm⁻¹, and 1098 cm⁻¹ regions confirm the presence of C-C stretching. The absorption bands in the 1081 cm⁻¹ and 703 cm⁻¹ regions indicate the presence of the benzene ring. The absorption band in the 1039 cm⁻¹ region is due to C-N stretching. The presence of absorption bands in the 948 cm⁻¹ and 641 cm⁻¹ regions is due to the 1,4-disubstitution pattern of the benzene ring. The absorption band in the 602 cm⁻¹ region is due to B-C deformation.

[0020] Chart C : The FT-IR spectrum of poly (N-isopropylacrylamide) is provided in this section. As can be seen, all characteristic absorption bands appear in their correct regions. The N-H stretching bands appear in the 3309 cm⁻¹ region, and the C-H stretching bands of the alkane chain appear in the 2876-2972 cm⁻¹ region. We see the C=O stretching bands in the 1723 cm⁻¹ region. The bands related to amide bonds appear in the 1541-1649 cm⁻¹ region, and the C-S stretching vibration appears in the 655 cm⁻¹ region.

[0021] Chart D : The FT-IR spectrum of the CSSU conjugate is provided in this section. As can be seen, the amide and carbonyl stretching bands appear in the 1651 cm⁻¹ and 1710 cm⁻¹ regions, respectively, and the characteristic bands related to the chitosan backbone appear in the 1080 cm⁻¹ region.

[0022]

[0023] Chart B: The ¹HNMR (CDCl3) spectrum of BPA-BOC is provided in this section. The presence of the BOC group is confirmed by a sharp peak in the 1.3 ppm region. A singlet peak corresponding to the nine hydrogens of the BOC₂O group appears in the 1.09 ppm region; the diastereotopic pattern of the phenylene group protons appears in the 2.8-3.0 ppm region. The presence of a singlet peak in the 4.1 ppm region indicates the N-H proton. In the 7.1 ppm and 7.6 ppm regions, two doublets corresponding to the five hydrogens of the aromatic ring appear, and the singlet peak appearing in the 8.26 ppm region confirms the presence of the hydroxyl group proton.

[0024] Chart C: The ¹HNMR (CDCl3) spectrum of poly (N-isopropylacrylamide) is provided in this section. According to the spectrum, two singlet peaks in the 1.1 ppm and 4.0 ppm regions correspond to the CH₃ and CH groups, respectively. Two relatively broad peaks between the 1.3 ppm and 2.1 ppm regions can be attributed to the CH and CH₂ groups in the main polymer chain. A broad and small peak in the 6.3 ppm region corresponds to the N-H proton. The carboxyl group proton appears in the 12 ppm region. All spectral evidence has confirmed the correct synthesis of the polymer.

[0025] Chart D: According to the ¹HNMR (D2O) spectra of the CSSU conjugate in this section, the characteristic peaks of chitosan appear in the 1.7-2.7 ppm and 3.0-4.0 ppm regions. We have two triplet peaks with equal intensity in the 2.1 ppm and 2.6 ppm regions related to the succinic anhydride groups. This spectral evidence confirmed the correct synthesis of the CSSU conjugate. The degree of substitution of the succinic anhydride group onto the chitosan backbone was calculated by considering the characteristic chitosan peaks in the 3-4 ppm region and the characteristic triplet peak of succinic anhydride in the 2.1 ppm region, and was found to be 21%.

[0026]

[0027] Figure C: In this section, the synthesis pathway of CSSU is provided. According to the schematic diagram, the resulting conjugate is formed through the reaction of the amine group of chitosan with the carboxyl group of succinic anhydride via amide bond formation.

[0028] Figure D: In this section, the synthesis pathway of the CSSU-PNI conjugate is provided. As can be seen, the resulting conjugate is formed by an amidation reaction between the carboxyl groups of poly (N-isopropylacrylamide) and the free amine groups of the chitosan backbone. During this reaction, the carboxyl group of the polymer is activated by EDC / NHS.

[0029] Figure E: In this section, the synthesis pathway of the CSSU-PNI-FPBA@BPA conjugate is provided. As is visible from the schematic diagram, the amidation of the carboxylic acid group of BPA-BOC with the free amine groups of the chitosan backbone was performed using carbodiimide coupling.

[0030]

[0031] Chart A : The FT-IR spectrum of the CSSU-PNI conjugate is provided in this section. As is observable in the spectrum and by comparing these spectra with the spectra of PNI and CSSU, the PNI and CSSU units are clearly visible. The vibrational bands appearing in the region above 3200 cm⁻¹ correspond to the N-H and O-H bands in the CSSU conjugate. The aliphatic C-H stretching bands of the alkane node in PNI appear in the 2875-2973 cm⁻¹ region. The C=O stretching bands of the carboxylic acid group and the amide bonds appear at 1651 cm⁻¹ and 1718 cm⁻¹, respectively. Finally, the characteristic bands of the chitosan backbone are observed in the 1072 cm⁻¹ region. An increase in the intensity of the amide bonds compared to the same bands in CSSU, as well as a decrease in the ratio of O-H and N-H bands to C-O-C, has confirmed the conjugation of PNI to CSSU.

[0032] Chart B: As observed in the ¹HNMR (D2O / DMSO) spectrum of the CSSU-PNI conjugate in this section, this conjugate was well-confirmed by comparing the CSSU-PNI spectrum with the spectra of CSSU and PNI. According to the spectrum for systems 1 and 2, all peaks related to CSSU and PNI appear in the expected regions. According to the spectrum, the peaks in the 1.98, 2.3, 2.6, 2.98 ppm regions in CSSU-PNI were compared with the peaks in the 3-3.8 ppm region in CSSU and the peaks appearing in the 1.2, 1.6, 2.1 ppm and around the 4.0 ppm regions corresponding to PNI. The molar ratio of conjugated PNI to CSSU, using the molecular weight of chitosan and PNI and the intensity of the related peaks at 1.16 ppm for PNI and 1.98 for CSSU and the calculations performed, was approximately 34 and 18 for the CSSU-PNI2 and CSSU-PNI1 systems, respectively.

[0033] Chart A: According to the FT-IR spectrum of the targeted systems 1 and 2, CSSU-PNI-FPBA@BPA, in this section, the vibrational bands in the 3440 cm⁻¹ region correspond to the stretching of N-H and O-H bonds, and the bands in the 2877-2971 cm⁻¹ region correspond to aliphatic C-H groups. In chitosan and PNI, the characteristic bands in the 1547, 1691, 1720 cm⁻¹ regions correspond to the C=O group stretching in the amide and acid bonds, respectively. The characteristic chitosan band corresponding to the asymmetric C-O-C stretching appears in the 1082 cm⁻¹ region. The characteristic bands related to BPA and FPBA (C-H of the phenyl ring) appear in the 3089 cm⁻¹ region and (B-O vibration) in the 874 cm⁻¹ region. We witness a relative increase in the intensity of the amide bonds and the ratio of the intensity of N-H and O-H bands compared to the C-O-C stretching bond due to the greater substitutions on the chitosan backbone.

[0034] Chart B: According to the ¹HNMR spectrum of the CSSU-PNI-FPBA@BPA systems 1 and 2, all characteristic peaks of PNI, CSSU, BPA-BOC, and FPBA have correctly appeared in the relevant regions. The specific peaks of BPA and FPBA in the aromatic region, a doublet in the 7.2-7.8 ppm region, and other related peaks have appeared in the 3 ppm and 4.1 ppm regions, which ultimately confirmed the successful synthesis of BPA@CSSU-PNI-FPBA. The degree of substitution of BPA and FPBA was calculated using the peak related to BPA and FPBA in the 7.2 ppm region and the peaks related to chitosan in the 3-3.8 ppm region. The percentage of FPBA attachment to CSSU-PNI for the CSSU-PNI2-FPBA and CSSU-PNI1-FPBA systems was approximately 22% and 15%, respectively. Also, the percentage of BPA attachment to CSSU-PNI-FPBA for the CSSU-PNI2-FPBA@BPA and CSSU-PNI1-FPBA@BPA systems was approximately 8.9% and 7.6%, respectively.

[0035] The attachment of BPA and FPBA to the chitosan backbone was confirmed using ¹¹B-NMR spectroscopy. As is observable in the spectrum, the peak that appeared in the 24 ppm range corresponds to B(OH)₂ that is attached to the phenyl ring.

[0036] The results from measuring the molecular weight of the polymer using the GPC technique are provided in this section. As can be seen, the weight-average molecular weight of the polymer (Mw) was 2311 and the number-average molecular weight of the polymer (Mn) was 1615.

[0037] In this section, the morphology and particle size of the CSSU-PNI-FPBA@BPA nanoparticles were investigated using SEM and TEM techniques.

[0038] In this section, the cytotoxicity of the drug-free nanoparticles and nanoparticles containing methotrexate and boron phenylalanine on the U87MG and A-431 cell lines was investigated. The toxicity of nanoparticle systems 1 and 2 containing the drug was investigated on two cell lines, U87MG (with overexpression of sialic acid receptors) and A-431 (without overexpression of sialic acid receptors), at a temperature of 39.5-40°C, which is presented in the bar charts. First, the cytotoxicity of the drug-loaded nanoparticles at physiological temperature (37°C) was investigated. According to the result obtained in the bar chart, the percentage of cell viability was above 90% and 80% at the lowest concentration for non-targeted and targeted nanoparticles, respectively, and above 80% and 70% at higher concentrations for non-targeted and targeted nanoparticles, respectively. These results confirm the drug release from the nanoparticles as previously reported using UV-Vis. The drug-free nanoparticles showed no toxicity even at higher concentrations on the two aforementioned cell lines. As is observable in the charts, at the highest concentration (40 µg / mL), the targeted and non-targeted drug-carrier nanoparticles showed the highest toxicity on the two cell lines U87MG and A-431, and in other words, the lowest percentage of viability compared to the free drug. Toxicity on the U87MG cell line is greater than on the A-431 cell line. There was a significant difference between different concentrations for the four nanoparticles in the viability rate with the same incubation time of 48 hours in both cell lines (P < 0.05). A significant difference was also observed between the nanoparticles with the negative control and the free drug for all 4 concentrations in both cell lines (P < 0.05). The increase in toxicity at different concentrations of the drug-loaded nanoparticles was due to the significant accumulation of the nanoparticles inside the two cell lines following endocytosis, which led to significant drug release from the nanoparticles as a result of the increase in temperature and the acidic pH. According to the IC50 values provided in the table, the IC50 of the targeted nanoparticles was lower than that of the non-targeted nanoparticles, and the percentage of viability for nanoparticles 2 was lower compared to nanoparticles 1, which was due to the nanoparticle's greater response to the temperature increase as a result of the higher polymer content. The targeted nanoparticles had a lower IC50 on U87MG cells compared to A-431 cells at all four concentrations, which was due to the ability of the boron phenylalanine molecules to interact with sialic acid receptors on the cell surface. The IC50 of the drug-loaded nanoparticles was lower compared to the free drug on both cell lines, indicating that the nanocarriers had far greater effects than the free drug. The obtained results are due to the thermos- and pH-sensitivity of the drug-delivering nanoparticles, which led to better drug transport and consequently improved drug performance. The increased anti-cancer activity of methotrexate was due to the protection of the drug from hydrolysis and degradation following its loading into the nanoparticles, and the temperature and pH sensitivity of these nanoparticles led to high drug release inside the tumor cells. Several pathways could have caused the increased anti-cancer activity of methotrexate loaded into the temperature- and pH-sensitive nanoparticles:

[0039] 1) Due to the increased endocytosis activity of tumor cells, the penetration of nanoparticles into the cells, as well as higher concentrations of methotrexate, increased.

[0040] 2) An increased concentration gradient near the surface of the tumor cells created a positive flux of nanoparticles into the cells.

[0041] 3) The boron phenylalanine molecules on the surface of the nanoparticles played an important role in the targeting of these nanoparticles; the more boron phenylalanine interacted with the sialic acid receptors on the surface of the tumor cells, the greater the passage through the cell membrane and the greater the toxic effects of methotrexate.

[0042] 4) Given that U87MG cells show resistance to common therapeutic methods including chemotherapy and anti-cancer drugs, nanoparticles with a particle size smaller than 200 nm have the ability to cross the cell membrane, thereby preventing cellular resistance with controlled drug release inside the cell. Studies conducted on methotrexate have provided evidence of the different toxicity of this drug on the two U87MG and A-431 cell lines.

[0043] As expected, the results obtained from the MTT assay show non-cytotoxicity of the nanoparticles containing boron phenylalanine drug and the boronated FPBA compound at all five concentrations on the U87MG cell line in the absence of thermal neutron irradiation. This is because, as mentioned at the beginning, the BPA drug and the boronated FPBA compound must undergo thermal neutron irradiation and the nuclear fission process and the release of alpha and lithium particles to exert their cytotoxic effects on the cell, a feature that is highly important in the BNCT therapeutic method.

[0044] In this section, the cellular uptake of the nanoparticles by the U87MG cell line was investigated using flow cytometry and fluorescence techniques. Cellular uptake was studied quantitatively and qualitatively by loading curcumin into the nanoparticles to evaluate the accumulation of nanoparticles inside tumor cells and also to prove that boron phenylalanine plays an important role in drug delivery into the cells. Due to the overexpression of sialic acid receptors on the surface of U87MG cells, nanoparticle endocytosis was enhanced by modifying the nanoparticle surface with BPA-BOC. According to the histogram plots and fluorescence images provided in the figure, the curcumin-loaded targeted nanoparticles in U87MG cells showed a significant increase in fluorescence intensity after two hours compared to the curcumin-loaded non-targeted nanoparticles, indicating that the attachment of boron phenylalanine to the chitosan backbone facilitated the cellular uptake of the targeted nanoparticles compared to the non-targeted nanoparticles. This difference between the cellular uptake of targeted and non-targeted nanoparticles was significant (P < 0.05), which was largely mediated by the interaction of boron phenylalanine with the sialic acid receptors that are overexpressed on the surface of U87MG cells.

[0045] According to the figure, and with the knowledge that the number of black dots, which are the result of thermal neutron collisions with the boron atoms loaded into the system and the resulting nuclear fission, is approximately the same for the BPA drug, FPBA, and the BPA@FPBA@CSSU-PNI2@BPA nanoparticle system, it can be proven that this thermos- and pH-sensitive drug delivery system has been able to demonstrate maximum efficiency in loading the boron-containing drug at concentrations similar to the boron phenylalanine drug.

[0046] According to the schematic diagram of the synthesis steps of the targeted CSSU-PNI-FPBA@BPA nanoparticles, due to the interaction between the positive charges of the chitosan backbone and the negative charges of the succinic anhydride groups attached to the NH₂ groups, the temperature-sensitive polymer N-isopropylacrylamide is located within the core, such that the chitosan-succinic anhydride plays the role of the shell. The pH-sensitive feature is also created through an imine bond following the conjugation of the boronated compound FPBA to the NH₂ groups of the chitosan backbone. Finally, the targeting of this nanoparticle occurs by conjugating BPA-BOC to the chitosan backbone. Under controlled chemical reactions, by activating the hydroxyl groups of the boronated compound FPBA, the formation of COF structures occurs through the growth of this compound following boroxine or boronic ester bonds. The loading of other anti-cancer drugs, such as methotrexate, in addition to the boronated drugs, is possible within these COF structures, and the BPA-targeted chitosan-poly(N-isopropylacrylamide) conjugate plays the role of a coating.

[0047] This table presents the results of a study conducted to demonstrate the change in the drug delivery system's behavior in environments with different pH levels. The most critical points include the retention of the drug at physiological pH (7.4) and its optimal release at acidic pH (e.g., 5.5) to correspond to tumor environments. The interpretation of this study indicates that at pH 7.4, the drug is effectively retained (proving the stability of the shell), whereas at pH 5.5, the FPBA–chitosan shell reacts (cleavage of the C=N bond), and rapid release occurs.

[0048] This table presents the results of a study that focuses on the effect of temperature changes (an increase in temperature past the lower critical solution temperature or LCST) on drug release. The primary objective is to prove that the PNIPAAm core is activated upon reaching the critical temperature (e.g., approximately 39 °C), leading to enhanced drug release. The interpretation of this study shows that a slight increase in temperature (LCST ≈ 39.5 °C) significantly accelerates release.

[0049] This table presents the results of a study designed to evaluate the performance of the drug delivery system for application in the Boron Neutron Capture Therapy (BNCT) method. In this method, the significance of boron (¹⁰B) loading into the nanoparticles and the production of α-particles upon neutron irradiation is investigated to prove the therapeutic efficacy of the drug delivery system. The interpretation of this study indicates:

[0050] High boron loading (≈ 30 µg / 10⁶ cells).

[0051] A very high number of α-tracks post-irradiation, indicative of effective fission and α-particle production.

[0052] A drastic reduction in clonogenic survival in the U87MG cell line (< 0.7%) and in the T98G cell line (~1.5%) in the group of nanoparticles carrying the two boronated drugs, compared to drug-free nanoparticles (> 75%). Therefore, it can be stated that nearly all U87MG cells were eliminated due to the toxicity of the boron atoms, and a significant difference was observable in the survival percentage of the two cell lines following thermal neutron irradiation and the induction of toxicity. In contrast, the survival percentage of the two cell lines exposed to the combination of the free drugs BPA and FPBA is significantly higher than that of the nanoparticles carrying the boronated drugs.

[0053] A- Protection of BPA with BOC₂O

[0054] 250 mg (2.392 mmol) of BPA was placed in a water bath at a temperature of 25°C under gentle stirring, together with 2.6 mL of 1 N sodium hydroxide (105 mg, 2.631 mmol), and 2 mL of tert-butyl alcohol was added thereto until the solution became diluted and clear. Subsequently, over a period of 1 hour, 0.54 mL (2.631 mmol) of BOC₂O was added dropwise to the solution until the solution became turbid. Upon completion of the dropwise addition of BOC₂O, the temperature was increased to 30-35°C and maintained for 30 minutes, and the mixture was subjected to vigorous stirring for 24 hours. After the said period, a white precipitate had formed at the bottom of the flask, which, according to the reference, had a pH of 8.35. The reaction mixture was extracted twice with 25 mL of cold pentane. The upper phase, which was the organic phase, was extracted four times with 10 mL of a saturated sodium bicarbonate solution. The pH of the lower phase, which was the aqueous phase, was adjusted to 1.65 by adding a potassium hydrogen sulfate solution. This phase was extracted four times with 40-mL portions of diethyl ether, washed twice with 20-mL portions of distilled water, and dried over anhydrous magnesium sulfate, which acts as a desiccant. The magnesium sulfate was separated by filtration, the diethyl ether was removed using a rotary evaporator, and a yellow oil remained at the bottom of the flask. It was dissolved in 20 mL of n-hexane, and crystal formation was induced by scratching the walls of the beaker and placing it in a freezer for 24 hours. After 24 hours, over the course of the day, 10×1, 4×5, and 2×2.5 mL of n-hexane were added to the formed crystals at the bottom of the beaker under stirring. It was then placed in the freezer for 24 hours, and white crystalline precipitates were formed. The precipitates were collected and washed with cold pentane. The reaction scheme is provided in Figure F-1, A.

[0055] B- Poly(N-isopropylacrylamide)

[0056] 2 g of NIPAAM monomer (17.67 mmol) and 50 mg of AIBN (0.573 mmol) were dissolved in 10 mL of isopropanol. Then, 50 µL (0.304 mmol) of 3-MPA (3-mercaptopropionic acid) was added, and the mixture was placed under nitrogen gas for 15 minutes and then placed in an oil bath at a temperature of 75°C under gentle stirring for 24 hours. After 24 hours and the completion of the polymerization reaction, diethyl ether was added to the product to induce precipitation. It was then placed in a rotary evaporator to remove the isopropanol, leaving a white powdery precipitate, which was our desired polymer. A portion of the polymer was dispersed in distilled water and dialyzed in a dialysis membrane with a cutoff of 2 kDa for 48 hours against deionized water, and finally lyophilized. The polymerization yield was calculated to be 96%. The reaction scheme is shown in Figure F-2, B. The structure of the resulting polymer was confirmed by FT-IR and ¹H-NMR techniques.

[0057] C- Succinylation of Chitosan with Medium Molecular Weight

[0058] At this stage, the purpose of succinylation was to increase the solubility of chitosan in water. Three different amounts (155, 77.5, 38.7) mg of succinic anhydride were used until suitable solubility was achieved. 1 mole of medium molecular weight chitosan with a degree of deacetylation of 76% and a monomeric unit mass of 177.17 has 974 free NH₂ groups. To 500 mg of chitosan, 20 mL of distilled water was added, and it was dispersed by placing it in a sonicator. Then, a 0.1% acetic acid-water solution was added dropwise to the chitosan under stirring, until the chitosan was completely dissolved by bringing the pH to 5.5, at which point it adopted a gel-like state. The chitosan solution was diluted by adding 20 mL of methanol. 155 mg of succinic anhydride was dissolved in 2 mL of acetone in a vial, and the solution was added via syringe to the chitosan solution and placed under nitrogen gas to render it free of air. It was then subjected to vigorous stirring at room temperature for 24 hours. After 24 hours and the completion of the reaction, the entire product was placed in a rotary evaporator with heat and vacuum to remove the methanol and acetone solvents. It was dialyzed in a dialysis membrane with a cutoff of 10 kDa for 48 hours against distilled water with a pH of 5.5-6, and after this period, it was lyophilized. The reaction scheme is provided in Figure F-3, C.

[0059] D- Synthesis of the Conjugate of Succinylated Chitosan with Poly(N-isopropylacrylamide)

[0060] First, to 200 mg and 400 mg of poly(N-isopropylacrylamide), a solution of EDC / NHS was added to activate the carboxyl groups of the polymer. The mixture was degased under nitrogen gas for 15 minutes and then under gentle stirring for 24 hours. After the required time and the activation of the poly(N-isopropylacrylamide), a 0.1% acetic acid-water solution (with a pH of 6-6.5) was added to 100 mg of succinylated chitosan, and it was sonicated for 15 minutes to dissolve completely. Then, the activated poly(N-isopropylacrylamide) solution was added to the above solution in ratios of (1:2 and 1:4), and it was subjected to gentle stirring for 24 hours. After the required time and the completion of the reaction, the clear-colored solution was dialyzed in a dialysis membrane with a cutoff of 10 kDa for 8 hours against ethanol, then for 24 hours against water with a pH of 7.4, and finally, it was lyophilized. The reaction scheme can be observed in Figure S-1, D. The structure of the sample was confirmed by FT-IR and ¹H-NMR techniques.

[0061] E- Synthesis of the Conjugate of Succinylated Chitosan-Poly(N-isopropylacrylamide) with the Boron-Containing Compound FPBA

[0062] E-1- Activation of FPBA

[0063] 200 mg (0.647 mmol) of FPBA was dissolved in 3.5 mL of DMSO anhydrous. Then, 120 mg of EDC (0.777 mmol) and 90 mg of NHS (0.777 mmol) in a 1.2 molar equivalent ratio were added to the FPBA solution, degased under nitrogen gas, and subjected to vigorous stirring for 24 hours.

[0064] E-2- Preparation of the Conjugate of FPBA with the CSSU-PNI System

[0065] 150 mg of the two CSSU-PNI conjugates were dispersed in a dilute acetic acid-water solution with a pH of 6. Then, under vigorous stirring, 1.5 to 2 mL of the clear, activated FPBA solution was added dropwise via syringe, along with nitrogen gas, to the CSSU-PNI system. During the addition, the solution became white and slightly viscous. The reaction was allowed to complete over a period of 24 hours. After the required time, the resulting solution was dialyzed in a dialysis membrane with a cutoff of 10 kDa for 6 hours against ethanol, then for 48 hours against water with a pH of 7.4, and finally, a lyophilized powder was obtained using a freeze-dryer. The reaction scheme is provided in Figure F-1, E. The structure of the system was confirmed by FT-IR and ¹H-NMR techniques.

[0066] F- Conjugation of BPA to the CSSU-PNI-FPBA System

[0067] F-1- Activation of BPA

[0068] 140 mg (0.453 mmol) of BPA-BOC was dissolved in 2.5 mL of DMSO anhydrous. Then, 84 mg of EDC (0.544 mmol) and 63 mg of NHS (0.544 mmol) in a 1.2 molar equivalent ratio were added to the BPA solution, degased under nitrogen gas, and subjected to vigorous stirring for 24 hours. The structure of the resulting compound was confirmed by FT-IR and ¹H-NMR techniques.

[0069] F-2- Preparation of the Conjugate of Activated BPA with the CSSU-PNI-FPBA System

[0070] 150 mg of the two CSSU-PNI-FPBA conjugates were dispersed in a dilute acetic acid-water solution with a pH of 6. Then, under vigorous stirring, 0.9 to 1 mL of the clear, activated BPA solution was added dropwise via syringe, along with nitrogen gas, to the CSSU-PNI-FPBA system. During the addition, the solution became white and slightly viscous. The reaction was allowed to complete over a period of 24 hours. After the required time, the resulting solution was dialyzed in a dialysis membrane with a cutoff of 10 kDa for 6 hours against ethanol, then for 48 hours against water with a pH of 7.4, and finally, a lyophilized powder was obtained using a freeze-dryer. The reaction scheme is provided in Figure F-1, F. The structure of the final system was confirmed by FT-IR and ¹H-NMR techniques.

[0071] G- Loading of the FPBA Compound into the CSSU-PNI-FPBA@BPA Targeted System

[0072] This was prepared using the solvent displacement / precipitation method. This method is based on dissolving the polymer in an organic solvent and the diffusion and displacement of the organic solvent into the aqueous solvent. In this method, 10 mg of FPBA and 20 mg of the system were dissolved in a minimum possible amount (7.5 mL) of a water-miscible organic solvent, such as the non-polar organic solvent DMSO. The solution was then injected forcefully and at regular intervals under sonication into 50 mL of deionized aqueous phase (a 1:10 ratio), whereupon loading occurs due to the diffusion of the water-miscible organic solvent into the aqueous phase. Finally, the aqueous solution containing the system carrying the FPBA compound was obtained as a lyophilized powder using a freeze-dryer.

[0073] H- Preparation of FPBA@CSS-PNI-FPBA@BPA Nanoparticles Containing Borophenylalanine, Methotrexate (MTX), and Curcumin (CUR) Drugs

[0074] Drug-containing chitosan nanoparticles were formed using the direct dialysis method. The method for preparing the nanoparticles is as follows: first, 10 mg of the two systems were dissolved in 5 mL of DMSO by placing them in a sonicator and under stirring. Then, in a 1:1 ratio, 10 mg of borophenylalanine (the primary boronated drug for BNCT studies), methotrexate (a model drug effective in glioblastoma treatment for assessing toxicity and nanoparticle efficacy), and curcumin (as a fluorescent drug for studying nanoparticle cellular uptake) were added to them. To achieve complete solubility, they were placed in a sonicator and then under stirring. The three drug-containing systems were placed in a dialysis membrane with a cutoff of 12 kDa for 24 hours against water with a pH of 7.4 under stirring, so that the organic solvent was removed and replaced by water, and the drug-containing nanoparticles were formed. During this period, the membrane environment was replaced every 4 hours. The drug loading amount in the systems was determined by examining the absorbance of free borophenylalanine, methotrexate, and curcumin in the external aqueous environment outside the membrane at a pH of 7.4, at maximum wavelengths of 260, 304, and 428 nm, respectively, using a UV-Vis spectrophotometer. Also, by centrifuging (15,000 rpm for 15 minutes) the drug-containing systems after removal from the membrane, the supernatant solution above the formed nanoparticles was examined by the UV-Vis spectrophotometer, and in both methods, no free drug was observed. Then, the drug-containing nanoparticles were lyophilized.

[0075] I- Determination of the LCST of the Two CSSU-PNI-FPBA@BPA Systems and the PNI Polymer

[0076] This test was performed using a UV spectrophotometer with a heating rate of 0.5 °C / min from a temperature of 25 to 50°C. The LCST (Lower Critical Solution Temperature)— a characteristic parameter for temperature-sensitive polymers—of the two systems and the polymer at concentrations of 2 mg / mL in phosphate buffer with a pH of 7.4 were measured, respectively. The effect of different polymer concentrations on the LCST is observable based on the obtained diagrams.

[0077] J- Effect of Different pH on the Release Rate of FPBA and Borophenylalanine Drug

[0078] This test was conducted utilizing the pH-sensitive property of the C=N double bond in the FPBA boron-containing compound attached to the chitosan backbone. 10 mg of the drug-containing nanoparticles were dispersed in a dialysis bag and immersed in 50 mL of phosphate buffer at two different pH values (7.4 and 5.5) as the dialysis medium and placed under stirring. pH monitoring was performed at time intervals of (0, 0.25, 0.5, 1, 2, 4, ..., 48) hours. At specified times, approximately 1 mL of the phosphate buffer containing the suspended drug was transferred into an Eppendorf tube, and the same amount of fresh phosphate buffer was replaced. The Eppendorf tubes were placed in a centrifuge at 15,000 rpm for 15 minutes so that the released drug in each tube was completely separated from the nanoparticles and located in the supernatant solution (phosphate buffer). The comparison of the release trend of the borophenylalanine drug from the core, which was incremental in the two dialysis media over the set time period, was investigated using an HPLC instrument. The comparison of the release trend of the FPBA drug from the shell in the two dialysis media over the set time period was investigated using a UV-visible spectrophotometer.

[0079] K- Neutron Irradiation Studies

[0080] K-1- Stage One: Neutron Capture by the Two Boron-Containing Compounds, BPA and FPBA, Loaded into the Shell and Core of FPBA@BPA@CSSU-PNI2-FPBA@BPA

[0081] The purpose of this stage is to investigate or measure the appropriate boron concentration to generate neutron irradiation products following thermal neutron flux. For this purpose, amounts of 30 µg of the BPA drug, FPBA, and the BPA@FPBA@CSSU-PNI2-FPBA@BPA system were spread relatively homogeneously onto CR-39 polymers with dimensions of 1×1 cm. Subsequently, the CR-39 polymers were exposed to a thermal neutron flux (5.6×10⁸ ncm⁻²s⁻¹) in the Tehran Research Reactor for 27 to 30 minutes. Then, the detection process was carried out on the thermal neutron-irradiated CR-39 polymers under chemical etching conditions. The chemical etching was performed with a 6.25 N NaOH solution at a temperature of 70°C (in an oven) for 5 hours.Examples

[0082] Upon confirmation of the non-toxicity of the temperature- and pH-sensitive nanoparticles in the absence of thermal neutron irradiation, and the results from neutron irradiation on the CR-39 samples, the quality and efficacy of the nanoparticles as boron carrier systems were established. Further evaluations are also underway to introduce a valuable and efficient temperature- and pH-sensitive drug delivery system for pre-clinical studies in BNCT. The establishment of suitable in vitro cytotoxicity on the U87MG cell line in the presence of neutron irradiation, which is currently underway, will confirm the efficacy of these boron phenylalanine-targeted, temperature- and pH-sensitive nanoparticles as boron carriers in the BNCT therapeutic method. Consequently, these nanoparticles can be utilized as boron carriers for the treatment of cancer cells, particularly glioblastoma, in vivo in the BNCT therapeutic method. Upon completion of the animal laboratory stages, these nanoparticles will acquire the potential for human application.

[0083] Currently, the BNCT therapeutic method in our country is stalled at the initial research stages, one of the reasons for which is the lack of boron-containing compounds or systems with applicability in this therapeutic method. In this research project, the synthesized boron-containing nanoparticles, with their inherent capability of loading and releasing a sufficient concentration of boron, can be employed as novel boron delivery systems in the BNCT therapeutic method—which is a complementary method for the treatment of cancers, particularly glioblastoma—and this therapeutic method itself can also be established in our country.

Claims

What is claimed is a pharmaceutical composition comprising boron-10 atoms, said composition being temperature- and pH-sensitive and having a core-shell structure, wherein the composition comprises:-a shell comprising a chitosan polymer, wherein said chitosan polymer is conjugated with boron phenylalanine and with boron-containing compounds such as formyl phenylboronic acid (FPBA), thereby imparting pH-sensitivity; and-a core comprising a temperature-sensitive polymer, poly(N-isopropylacrylamide), conjugated to the chitosan backbone, wherein said core and said shell interact with one another to maintain the integrity of the assembly external to a target cancer cell;wherein phenylalanine-containing compounds on the surface of the shell comprise boron phenylalanine, which is linked via an amide bond to free NH₂ groups of the succinylated chitosan backbone, and which binds to sialic acid receptors on the surface of the target cancer cell.A composition according to claim 1, wherein the compounds within the shell comprise poly(N-isopropylacrylamide), boron phenylalanine (BPA), and formyl phenylboronic acid (FPBA), and wherein the molar ratio of formyl phenylboronic acid to boron phenylalanine is determined according to experiments performed on glioblastoma cancer cells to establish the appropriate ratio for a desired synergistic effect that is dependent on the type of cancer cell. The set of said compounds within the shell is attached to a shell made of chitosan via attachment to NH₂ groups.A composition according to claim 1, wherein the temperature-sensitivity of said composition results from the linkage of the poly(N-isopropylacrylamide) polymer, and the pH-sensitivity results from an imine bond formed by the linkage of the formyl phenylboronic acid compound to the NH₂ groups of the chitosan backbone, wherein said compound can be a part of the shell and linked thereto, and also a part of the core and linked to the poly(N-isopropylacrylamide), which, when linked to the shell, provides a two-stage functionality for the drug delivery system, such that first the pH-sensitivity of the composition is activated, and subsequently the temperature-sensitivity of the composition causes the release of the boron-containing compounds.A composition according to claims 1 and 3, wherein the formation of a Covalent Organic Framework (COF) structure occurs following the formation of pH-sensitive boronate ester or boroxine bonds via the growable formyl phenylboronic acid compound under controlled reactions, wherein the chitosan-poly(N-isopropylacrylamide) conjugate can function as a coating on said COF structure, and wherein drug loading takes place in the coating and the core of the COF structure.A composition according to claim 1, wherein the size of the composition is 100-200 nanometers and the shape of the composition is substantially spherical.

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