bionanoprobes

Abstract

The invention relates to theranostic bionanoprobes capable of targeted, magnetic, pH and light sensitive, controlled drug release for the diagnosis and treatment of breast cancer.

Description

[0001] BIONANOPROBES

[0002] Technical Area

[0003] The invention relates to targeted, magnetic, pH, and light sensitive, controlled drug delivery theranostic bionanoprobes for the diagnosis and treatment of breast cancer.

[0004] State of Art

[0005] Diagnostic and therapeutic methods that play an active role in the fight against cancer, in recent years, with technological developments, new nanoformulations that combine diagnosis and treatment in a single material and enable targeted imaging, molecular therapy and clinical applications are being developed. Due to their low toxicity, ease of synthesis and modification, ability to be synthesized in different sizes and the potential to be combined with various imaging technologies, upconversion (improving image quality) luminescent nanoparticles (UCNP) come to the fore in theranostic applications.

[0006] UCNPs are lanthanide ion (Ln3+) based inorganic nanomaterials that emit higher energy visible light when excited with low energy NIR light. Compared to classical fluorophores, UCNPs have the advantages of minimal photodamage in living organisms, low autofluorescence, high signal-to-noise ratio and detection sensitivity, and high penetration depth in biological and environmental samples (Gu et al., 2013). Currently, these rare earth doped inorganic nanomaterials are considered to be non-toxic or low toxic to cells and tissues (Chatterjee et al., 2010). Studies have shown that the average effect of in vitro exposure to UCNPs in terms of cell viability does not exceed 5-20% of cell population loss. It has been found that UCNPs conjugated with chemotherapeutic agents exhibited the expected significant cytotoxicity in tumor cells (Chen et al., 2014; Min et al., 2014; Wang et al., 2011; Yang et al., 2014).

[0007] UCNP structures consist of rare earth elements (lanthanides) doped in a host matrix. Lanthanide-doped UCNPs can be excited by NIR light and emit light in a wide range of wavelengths ranging from UV to NIR. This is called upconversion and can be achieved by embedding Ln3+ions in a suitable inorganic host matrix using staircase-like energy levels. In UCNP synthesis, dopants such as Yb3+or Nd3+are preferred as sensitizer Ln3+ions to increase the NIR absorption power, while Er3+, Tm3+, Ho3+and Ln3+ions are used as activators (Lingeshwar et al., 2018). Lanthanides are generally placed in host matrices with chloride, bromide or fluoride ions. In order to increase the UC photoluminescence efficiency and reducenon-radioactive losses, the selected host materials are preferred to have low photon energy. Among these host materials, fluorides are ideal as additives in the preparation of UCNPs due to their generally low photon energy (~350 cm-1) and high chemical stability (Wang, et al., 2009). In addition, regardless of the concentration of the additive, the UC activity is significantly affected by the host crystal structure. For example, the green emission intensity of ^-NaYF4: Yb3+ / Er3+in the hexagonal phase is 10 times higher than that of a-NaYF4: Yb3+ / Er3+in the cubic phase.

[0008] After synthesis, the obtained UCNPs are generally hydrophobic because of oleic acid coating. There are various surface modification strategies to increase stability and change surface polarity under physiological conditions. The simplest of these is the oxidation of the double bond in oleic acid to epoxide or carboxy groups (Muhr et al., 2014). Another method is to form a double layer with a hydrophilic group by mixing with phospholipids or amphiphilic molecules such as Tween 80 (Muhr et al., 2014). The ligand exchange method, in which oleic acid is replaced with another molecule that binds more strongly to the surface (polymers with carboxylic or amine / phosphate functionalities), is widely used and is a preferred method for polymer coating. In a study, polyvinylpyrrolidone) coated UCNPs synthesized by the hydrothermal method were synthesized and then coated with poly(ethylamine) and poly(acrylic acid) by ligand exchange reaction. According to the results of the study, it was stated that UCNPs coated with poly(ethylamine) are the most suitable structure for cellular uptake due to their positive charge. Other surface modification methods are silanization and layer formation methods (Liu et al., 2013). In the silanization method, the UCNP surface is coated with a silica layer to obtain an inorganic surface. With this coating, the luminescence intensity of UCNPs is not reduced, while the toxic effect is minimized and a hydrophilic structure is gained.

[0009] While MRI, one of the clinical imaging methods, provides three-dimensional, high-resolution photographs compared to other imaging techniques, it causes poor detection sensitivity in the early imaging of pathological tissues or in cases where sufficient tissue separation cannot be made (Artemov et al., 2003). Contrast agents are needed to overcome this limitation. The change in the relaxation time in tissues containing these substances increases the contrast and makes the tissue more distinct. Today, iron oxide nanoparticles can be used in MRI imaging. In general, the lack of tissue selectivity of these substances, their short-term presence in the vascular system and the toxicity of metal ions limit the clinical applications of these substances. Therefore, the search for an ideal nanoparticle-based contrast agent that provides a moreeffective image at a lower dose, significantly reduces the imaging time, shows less toxicity, can remain in the endosomal-lysosomal cycle for a longer time, is biocompatible, biodegradable and targetable has become a subject that scientists have focused on. For biomedical applications, particle size control, surface chemistry and particle stability are important. The methods used to control particle size are to coat magnetic particles with organic molecules to ensure stability. On the other hand, for selective interactions, it is of great importance to bind the target molecule to the nanoparticle surface without disrupting its structure during surface coating. Two important parameters must be taken into account to develop an effective target contrast agent. First, appropriate molecular markers that are found in small amounts in normal tissues but abundant in diseased tissues must be selected. Second, the selected molecular agents must have high binding affinity at the targeted site.

[0010] Many drug delivery systems have been developed to date. Advances in polymer chemistry and the development of new polymerization techniques have enabled the synthesis of polymers with well-defined narrow molecular weight distributions that can alter their structural and physical properties in response to environmental factors such as heat, light, pH, and sonication (Carraher et al., 2013; Braunecker et al., 2007; Moad et al., 2008). Combined with progress in molecular cell biology, these advancements have facilitated the design of sophisticated, selective, and targeted nanodrug carriers containing biologically active compounds, such as active pharmaceutical ingredients, genes, enzymes, other proteins, or nucleotides. These nanocarrier systems can be used as highly specialized, safe, and effective agents for cancer treatment. Lin and colleagues synthesized upconversion nanoparticles (UCNPs) using the thermal decomposition method and initially coated them with poly(acrylic acid) (PAA) via ligand exchange, followed by coating with poly(ethylenimine) (PEI). Subsequently, they complexed the UCNP / PAA / PEI with MDR1 -siRNA, which suppresses the MDR-1 gene, a key factor in multi drug resistance. The study demonstrated that the UCNP / PAA / PEI / MDR1 -siRNA nanocomplex enabled effective gene silencing and resensitized paclitaxel-resistant ovarian cancer cells to paclitaxel treatment (Lin et al., 2017). Another advantage of using polymers and nanoparticles as drug delivery systems is the increased solubility and bioavailability of hydrophobic drugs in water, prolonged circulation time in the blood, protection of the active ingredient from inactivation in biological environments, and the ability to specifically target tumor cells (Reddy et al., 2010; Hillery et al., 2001; Devajaran et al., 2012; Elsabahy et al., 2012; Delplace et al., 2014; Nicolas et al., 2013). Controlled activation / deactivation of drugrelease prevents toxicity or other side effects in healthy cells and tissues. Biopolymers, particularly sugars and proteins, have shown promising results in drug delivery applications due to their biocompatibility and biodegradability (Hawkins et al., 2008; Liu et al., 2013; Chen et al., 2012; Wu et al., 2009; Roy et al., 2003). Poly(acrylic acid) (PAA) is the most commonly used pH-sensitive hydrophilic polymer for coating UCNPs. After coating UCNPs via ligand exchange, PAA chains not only provide hydrophilicity to the structure but also offer abundant carboxyl groups for drug loading through electrostatic interactions. Studies have shown that the release rate and amount of Doxorubicin (Dox) from PAA-coated UCNPs are significantly higher at pH 5.0 compared to pH 7.4. This pH-sensitive release is attributed to the protonation of carboxyl groups in PAA at low pH, weakening the electrostatic interaction between PAA and Dox. In contrast, at neutral pH (pH = 7.4), PAA is negatively charged and strongly binds to positively charged Dox. Here, UCNPs serve as monitors providing feedback on drug release when used with fluorescent drugs like Dox. Using two-photon laser scanning microscopy, upconversion signals from UCNPs under 980 nm excitation and fluorescence signals from Dox under 488 nm excitation can be simultaneously obtained. The 550 nm emission of UCNPs matches well with Dox absorption, enabling the monitoring of drug release behavior (Jia et al., 2013; Bei et al., 2015). In their study, Chen and colleagues developed a Trojan horse-like amphiphilic nanoparticle by coating UCNPs with PAA and polydopamine for pH / NIR dual therapy. They then loaded this nanoparticle with the hydrophilic drug Dox and the hydrophobic drug sorafenib, enabling the release of the drugs under pH / NIR dual stimuli for the diagnosis and prevention of hepatocellular carcinoma metastasis (Chen et al., 2021).

[0011] The entry and exit of molecules into and out of cells are facilitated by specific proteins called glucose transporters (GLUT) (Zhao et al., 2007; Mueckler et al., 2013). To date, fourteen glucose transporter proteins have been identified. Among these, GLUT5 is a specific fructose transporter (McQuade et al., 2013). Recent research has shown that GLUT5 is overexpressed in breast cancer cells, while it is present at normal levels in healthy breast tissue (Szablewski, 2013; Barron et al., 2016). Godoy and colleagues also demonstrated the presence of GLUT5 in human breast cancer (Godoy et al., 2006). These findings indicate that human breast cancer cells can be characterized by their fructose transport capacity. This suggests that a high-affinity fructose transporter could provide new opportunities for developing early diagnosis and treatment strategies for breast cancer. Based on this idea, in a study we published, we demonstrated that fructose-coated polymeric micelles could be used as targeted drug deliverysystems in triple-negative breast cancer (TNBC) (Zhao et al., 2014; Dag et al., 2016). Additionally, fructose-substituted phosphorescent metal complexes have been tested in vitro as imaging agents for breast cancer, and their efficacy has been demonstrated (Zhang et al., 2013; Lo et al., 2013). Similarly, fructose molecules derivatized with fluorescent compounds have been used as imaging agents in MDA-MB-435, MDA-MB-231, and MCF7 breast cancer cells (Levi et al., 2007). Based on the knowledge gained from our previous studies, fructose can be used as a targeting agent due to its high binding affinity to relevant receptors in breast cancer. UCNPs can be combined with other metallic nanoparticles such as Au nanoparticles (Cai et al., 2017), CuS NPs (Su et al., 2017), ZnO NPs (Wang et al., 2015), TiO2 NPs (Tong et al., 2017), and superparamagnetic Fe3O4 (Zhang et al., 2012) to act as multifunctional "theranostic" platforms in a single hybrid system, offering new possibilities for targeted drug delivery. Among various nanomaterials, the combination of UCNPs and Fe3O4 NPs is particularly noteworthy as it provides multimodal imaging capabilities by utilizing both UCL and magnetic properties together. Additionally, this combination holds potential for targeted drug delivery and use as a photothermal therapy agent. The easiest way to encapsulate Fe3O4 NPs with UCNPs is to load Fe3O4 NPs into the porous structure of mSiO2 (Liu et al., 2015). Another method for encapsulating hydrophobic UCNPs with iron oxide nanoparticles (IONPS) is to use an amphiphilic block copolymer to obtain multifunctional micelles (Xu et al., 2011).

[0012] Resistance to chemotherapeutic agents in cancer, despite the frequent use of combination therapies, often leads to treatment failure (Gottesman et al., 2002; Jabr-Milane et al., 2008). Today, numerous cancer genes regulating apoptosis, proliferation, cell signaling, and multidrug resistance (MDR) have been identified (Shum et al., 2016). Gene therapy, specifically targeting these genes, either alone or in combination with chemotherapy, is an area of significant research interest (Benoit et al., 2010; Deng et al., 2013). Genetic materials like siRNA and DNA are utilized to selectively silence target genes such as Bcl-2 for antiapoptotic effects (Lima et al., 2004), mdrl for drug resistance (Pichler et al., 2005; Wu et al., 2003), and rad51 / 50 for drug sensitization (Hannay et al., 2007). Various studies have shown promising results in silencing these genes (Kang et al., 2007; Lillig et al., 2004; Liu et al., 2004; Suzuki et al., 1998). For instance, delivery of Dox and Bcl-2 siRNA to target sites via dendrimer-modified mesoporous silica nanoparticles resulted in significantly increased tumor cell apoptosis compared to separate administration of Dox and Bcl-2 siRNA (Chen et al., 2009). Similarly, combining low-dose doxorubicin and cisplatin with a BH-3 mimetic drug effectively overcame chemotherapy resistance and sensitized cancer cells to chemotherapeutic agents (van Oosterwijk et al., 2012).In another study, delivery of Bcl-2 siRNA and Dox via a chitosan-based carrier system demonstrated increased tumor inhibition both in vitro and in vivo (Yan et al., 2020). However, systemic administration of siRNA is limited by rapid degradation by RNases in the bloodstream and renal filtration due to their size (<6 nm), which reduces their therapeutic efficacy (Shum et al., 2016). To address this, carrier vector systems capable of transferring genetic material into cells have been explored. Viral vectors efficiently deliver genetic material into targeted cells by evading the immune system (Yoo et al., 2011). Despite their optimization, viral vectors face safety and immunogenicity challenges. Researchers have attempted to mimic viruses with synthetic polymers, modifying them with polysaccharides, proteins, and RNA to enhance their efficacy on cancer cells (Yoo et al., 2011). Compared to viral vectors, non-viral synthetic gene delivery vectors exhibit great advantages. In order for these synthetic non-viral vectors to be defined as an ideal gene delivery platform, they should have high gene delivery capacity, be biocompatible, and be resistant to serum proteins and targetable. (Gary et al., 2007) There is still a great need for the design, development, production and clinical testing of vectors with low cytotoxicity and high gene transfection efficiency, and for new generation siRNA carriers for a successful treatment. The most important parameter for an effective and successful delivery system is a stable complexation. For this purpose, parameters such as hydrophobicity / hydrophilicity ratio, polymer molecular weight, and charge density can be adjusted to obtain an optimum complexation. N / P ratio (protonable amine group in the polymer / phosphate group in the nucleic acid), pH, and ionic strength can affect the electrostatic bonding between the genetic material and the cationic polymer. Therefore, it has become a natural necessity to produce methods to prepare cationic synthetic gene carrier vectors with a well-defined architecture, known molecular weight, and structure. It is thought that this gap can be filled by producing synthetic cationic glycopolymers that have controllable molecular weight and structure, are biocompatible and biodegradable.

[0013] Figures

[0014] Figure A: Visual representation of in vivo model establishment and monitoring stages of the treatment group.

[0015] Figure B: Visual representation of the production stages of the nanoprobe subject of the invention.

[0016] Figure 1: DLS results of the prepared Fe₃O₄ nanoparticles.

[0017] Figure 2: DLS analysis results of the prepared Fe₃O₄ nanoparticles.

[0018] Figure 3: HR-TEM image of the prepared Fe₃O₄ nanoparticles.

[0019] Figure 4: DLS and HR-TEM analysis results of the prepared UCNP nanocrystals.Figure 5: DLS and TEM analysis results of the prepared UCNP nanocrystals after ligand exchange.

[0020] Figure 6: EDS results of the prepared UCNP nanocrystals.

[0021] Figure 7: Analysis results of the prepared UCNP -based nanoprobes and their precursors: A) Zeta potential, B) Photoluminescence, C) TGA, D) XRD.

[0022] Figure 8: A) ¹H-NMR and B) ¹³C-NMR spectrum of the 1-azido-3-amino propane compound (CDCl₃).

[0023] Figure 9: FT-IR spectra of 3-bromo-propanamine.HCl (black) and 1-azido-3-amino propane (red) compounds.

[0024] Figure 10: ¹H-NMR and ¹³C-NMR spectra (CDCl₃) of trimethylsilyl-protected alkyne-functionalized 4-cyanopentanoic acid dithiobenzoate (TSA-CPADB) RAFT agent and its precursor compounds.

[0025] Figure 11: FT-IR spectra of trimethylsilyl-protected alkyne-functionalized 4-cyanopentanoic acid dithiobenzoate (TSA-CPADB) RAFT agent and its precursor compound.

[0026] Figure 12: A) ¹H-NMR and ¹³C-NMR spectrum (CDCl₃) of 2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (iprFr) compound.

[0027] Figure 13: A) ¹H-NMR and B) ¹³C-NMR spectrum (CDCl₃) of 1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (iprFrMA) monomer.

[0028] Figure 14: FT-IR spectra of 1 -O-methacryloyl-2, 3:4,5-di-O-isopropylidene-P-D-fructo pyranose (iprFrMA) compounds and their precursors.

[0029] Figure 15: A) ¹H-NMR and B) ¹³C-NMR spectra (CDCl₃) of N-(3,4-dihydroxyphenethyl)methacrylamide (DopMA) monomer.

[0030] Figure 16: Comparison of FT-IR spectra of N-(3,4-dihydroxyphenethyl)methacrylamide (DopMA) monomer and its precursor compound.

[0031] Figure 17: A) ¹H-NMR and B) ¹³C-NMR spectra (CDCl₃) of 2-(furan-2-yl)-1,3-dioxolane (FurKet) compound.

[0032] Figure 18: A) ¹H-NMR and B) ¹³C-NMR spectra (CDCl₃) of (3aS,4R,7R,7aR)-4-(1,3-dioxolan-2-yl)-3a,4,7,7a-tetrahydro-4,7-epoxyisobenzofuran-1,3-dione (FurKetMi) compound.

[0033] Figure 19: A) ¹H-NMR and B) ¹³C-NMR spectra (CDCl₃) of (3aS,4R,7R,7aR)-4-(1,3-dioxolan-2-yl)-2-(2-hydroxyethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-1,3(2H)-dione (FurKetMiOH) compound.

[0034] Figure 20: A) ¹H-NMR and B) ¹³C-NMR spectra (CDCl₃) of 2-((3aS,4R,7R,7aR)-4-(1,3-dioxolan-2-yl)-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl methacrylate (MiMA) compound.

[0035] Figure 21: FT-IR spectra of MiMA monomer and its precursor compounds FurKet, FurKetMi, and FurKetMiOH.

[0036] Figure 22: A) ¹H-NMR and B) ¹³C-NMR spectrum (CDCl₃) of N-(tert-butoxycarbonyl)-L-lysine N-carboxyanhydride (tBoc-Lys-NCA) compound.

[0037] Figure 23: FT-IR spectra of ZBoc-Lys and ZBoc-Lys-NCA.

[0038] Figure 24: Analysis results of P(tBoc-Lys)-N₃ polymer: A) THF-GPC, B) FT-IR, and C) ¹H-NMR.

[0039] Figure 25: Analysis results of prepared PMiMA homopolymer: A) THF-GPC and B) ¹H-NMR.

[0040] Figure 26: Analysis results of prepared P(MiMA-b-DopMA) diblock copolymer: A) THF-GPC and B) ¹H-NMR.

[0041] Figure 27: Analysis results of P(MiMA-b-DopMA-b-iprFrMA) polymer: A) THF-GPC, B)1H-NMR, and C) FT-IR.

[0042] Figure 28: Analysis results of P(MiMA-b-DopMA-b-iprFrMA) triblock copolymer after TBAF hydrolysis: A) THF-GPC and B) ¹H-NMR.

[0043] Figure 29: Analysis results of P(MiMA-b-DopMA-b-iprFrMA)-b-P(tBoc-Lys) polymer: A) DMF-GPC and B) ¹H-NMR.

[0044] Figure 30: Analysis results of P(MiMA-b-DopMA-b-FrMA)-b-P(Lys) polymer: A) THF-GPC, B) ¹H-NMR, and C) FT-IR.

[0045] Figure 31: Analysis results of P(MiMA-b-DopMA-b-FrMA)-b-P(Lys) / Dox polymer: A) THF-GPC and B) ¹H-NMR.

[0046] Figure 32: DLS analysis results of prepared UCNP@MP-Dox nanoparticles and their precursors.

[0047] Figure 33: HR-TEM images of prepared UCNP@P-Dox and UCNP@MP-Dox nanoparticles.

[0048] Figure 34: A) Photothermal efficiency of UCNP@MP-Dox solution at different laser intensities and B) different concentrations. C) Photothermal efficiency of UCNP-C, Fe₃O₄, P-Dox, UCNP@MP-Dox, and water under laser irradiation. D) Linear correlation between the negative natural logarithm of 0 and time ( / ) obtained from the cooling period. E) UV-Vis-NIR spectrum of UCNP@MP-Dox. F) Photothermal stability test of UCNP@MP-Dox during five cycles of repeated laser irradiation.

[0049] Figure 35: A) Hysteresis loop of Fe₃O₄ and UCNP@MP-Dox. B) Graph of 1 / T₂ values at various iron concentrations.

[0050] Figure 36: Dox release profile graphs of A) P(MiMA-Z>-DopMA-Z>-FrMA)-Z>-P(Lyz) / Dox polymer, B) UCNP@P-Dox, C)UCNP@MP-Dox nanoparticles and D) UCNP@MP-Dox nanoparticles after NIR excitation.

[0051] Figure 37: UCNP@P-Dox / siRNA carrying capacity, all ratios are expressed as GNP / siRNA ratio (GNP: UCNP@MP).

[0052] Figure 38. siRNA and UCNP@MP- Dox and siRNA DLS analysis results.

[0053] Figure 39: Release measurement of UCNP@MP-Dox nanoparticle carrying siRNA.

[0054] Figure 40: Effect of UCNP, P and P-Dox on cell viability in MCF7 and 3T3 cells after 24 and 48 hours of incubation.

[0055] Figure 41: Effects of P-Dox, P-Dox / siRNA, UCNP@P-Dox, UCNP@P-Dox / siRNA, UCNP@MP-Dox and UCNP@MP -Dox / siRNA on cell viability in MCF7 and 3T3 cells after 24 hours of incubation with / without NIR stimulation.

[0056] Figure 42: Effect of P-Dox, P-Dox / siRNA, UCNP@P-Dox, UCNP@P-Dox / siRNA, UCNP@MP-Dox and UCNP@MP -Dox / siRNA on cell viability after 48 hours of incubation with / without NIR stimulation in MCF7 and 3T3 cells.

[0057] Figure 43: Changes in cell uptake amounts of Dox, UCNP, P and UCNP@P-Dox of 3T3 / NIH cells at 6, 12 and 24 hours. (Black line represents the control group, Green line represents 3.9 p / mL, Blue line represents 7.8 pg / mL and Red line represents 15.6 pg / mL doses).

[0058] Figure 44: Changes in cell uptake amounts of Dox, UCNP, P and UCNP@P-Dox of MCF7 cells at 6, 12 and 24 hours. (Black line represents the control group, Green line represents 3.9 p / mL, Blue line represents 7.8 pg / mL and Red line represents 15.6 pg / mL doses).

[0059] Figure 45: Cellular uptake efficiencies of UCNP @P -Dox / siRNA in MCF7 and 3T3 cells at 12 and 24 hours.

[0060] Figure 46: Comparison of UCNP, Dox and UCNP@P-Dox / siRNA cellular uptake efficiencies in MCF7 and 3T3 cells at a) 6th, b) 12th, and c) 24th hours.

[0061] Figure 47: UCNP and Dox uptake activities of MCF7 and 3T3 cells at 12 and 24 hours.

[0062] Figure 48: Apoptosis analysis of MCF7 and 3T3 cells.Figure 49: BCL 2, BCL-x, MCL1, BAD, BAX, NOXA mRNA expression levels in MCF 7 cells.

[0063] Figure 50: IVIS image showing the biodistribution of Dox and UCNP@P -Dox / siRNA groups at 0, 3, 6, 9, 12, and 24 hours after injection.

[0064] Figure 51: Tracking graph of weight and tumor volume in eight different treatment groups. The black arrow indicates the day of average tumor onset.

[0065] Figure 52: A) Ex vivo fluorescence images recorded 24 hours after injection of Dox and UCNP@P-Dox / siRNA in tumor-bearing mice, and B) graph showing their distribution in major organs and tumor tissues and their fluorescence signal intensities (KC; abbreviation for liver, AC; abbreviation for lung) (*p<0.05).

[0066] Figure 53: Effect of eight different treatment groups on tumor size.

[0067] Figure 54: H& E (arrowhead: mitotic figures, arrow: tumor cell, star: fibrous tissue formation), PCNA, Bcl-2, and Bax immunohistochemical staining images (arrowhead: intranuclear immunopositive tumor cells, star: fibrous tissue formation) of MCF-7 tumor sections from different treatment groups on day 24 (scale bar: 50 pm).

[0068] Figure 55: Double immunofluorescence staining of tumor, Dox, UCNP@P-Dox and UCNP@P-Dox / siRNA groups. Bcl-2 immunofluorescence (FITC), BAX immunofluorescence (Texas red), nuclear immunofluorescence staining (DAPI), merged Bcl-2, BAX, and nuclear staining (scale bar: 50 pm).

[0069] Figure 56: Statistical values of Bcl-2 and BAX immunoreactivity scores among four groups (***p<0.001, ****p<0.0001).

[0070] Detailed Description of the Invention

[0071] In the invention, a new generation biocompatible nanotherapeutic platform was developed, which was directed to target cancer cells with sugar molecules, complexed with anti-Bcl-2 siRNA to reduce the anti-apoptotic resistance of the cells, and which also enables diagnosis by using magnetic resonance imaging (MRI) and bioluminescence techniques together, as well as controlled drug release, and its effectiveness on breast cancer was examined in vitro and in vivo.

[0072] Synthesis of Initiators and Monomers

[0073] Synthesis of l-Azido-3- Aminopropane for Use in Ring-Opening Polymerization

[0074] The compound l-azido-3 -aminopropane, to be used as an initiator in the ring-opening polymerization reaction, was synthesized through a substitution reaction. 3-Bromopropylamine. HCl (1 eq.) and sodium azide (3 eq.) were dissolved in deionized water and stirred overnight at 80 °C. The next day, the solvent was removed under vacuum, and the mixture was extracted three times with diethyl ether. After drying over MgSO4, the solvent was removed under vacuum, and the product was purified by vacuum distillation. The product was obtained with a yield of 75% and stored at -20 °C.1) NaN3, H20, 80 °C

[0075] Br NH2.HBr → N3NH2

[0076]

[0077] 2) KOH, 10 °C

[0078] Scheme 1. Synthesis of l-azido-3 -amino propane.

[0079] The obtained product was characterized using 'H-NMR spectroscopy and FTIR analyses. According to the 'H-NMR spectrum in Figure 8A, the shift of the methylene protons attached to the azide group from 3.9 ppm to 3.4 ppm confirms the successful synthesis of the compound. Additionally, Figure 9 provides a comparison of the FTIR spectra of l-azido-3 -aminopropane and its precursor compound, 3-bromopropylamine. HCl. In the FTIR spectrum of the 1-azido-3 -aminopropane compound, the appearance of the absorption peak for the azide group at ~2090 cm-1and the absorption peak for the amine group at ~3500 cm-1indicates that the substitution reaction was successfully carried out.

[0080] Synthesis of the RAFT Agent

[0081] The synthesis of the trimethylsilyl-protected alkyne-functionalized 4-cyanopentanoic acid dithiobenzoate (TSA-CPADB) RAFT agent was carried out in three stages. First, the precursor compound di(thiobenzoyl)disulfide was synthesized, and the chemical structure of the obtained product was characterized using 'H-NMR analysis (Figure 10A). Upon examining the 'H-NMR spectrum provided in Figure 10, the peaks corresponding to the phenyl protons in the structure of the di(thiobenzoyl)disulfide compound appear between 7.20-8.15 ppm. In light of these results, the first precursor compound, di(thiobenzoyl)disulfide, was successfully synthesized for the RAFT agent.

[0082] S, CS2

[0083]

[0084] NaOCH3, metanol Scheme 2. Synthesis of di(thiobenzoyl) disulfide.

[0085] In the second step of the RAFT agent synthesis within the scope of the project, the compound synthesized in the first step was reacted with 4,4'-azobis(4-cyano pentanoic acid), resulting in the formation of the 4-cyanopentanoic acid dithiobenzoate (CPADB) compound.o o

[0086] EtOAc

[0087]

[0088] Scheme 3. Synthesis of 4-cyanopentanoic acid dithiobenzoate (CPADB).

[0089] The CPADB compound was first isolated using column chromatography with an ethyl acetate: hexane (1:4) eluent system. The isolated pink solid product was purified by crystallization in an ethyl acetate: hexane (1:3, v / v) mixture. To elucidate the structure of the synthesized CPADB compound, the ¹H-NMR spectrum provided in Figure 10B was examined. The peaks corresponding to the -CH2CH2 group protons, labeled (b) and (c), appeared at 2.45-2.73 ppm, while the peaks for the methyl group (-CH₃) and phenyl ring protons were observed at 1.95 ppm and 7.40-7.90 ppm, respectively. Based on these results, the CPADB compound was successfully synthesized.

[0090] In the third step of the RAFT agent synthesis, CPADB reacted with trimethyl silyl propargyl alcohol to yield the trimethylsilyl-protected alkyne-functionalized 4-cyanopentanoic acid dithiobenzoate (TSA-CPADB) RAFT agent.

[0091]

[0092] Scheme 4. Synthesis of trimethylsilyl-protected alkyne-functionalized 4- cyanopentanoic acid dithiobenzoate (TSA-CPADB) RAFT agent.

[0093] The chemical structure of the product was characterized using ¹H-NMR spectroscopy. Upon examining the ¹H-NMR spectrum of the TSA-CPADB compound in Figure 10D, the methylene protons appear at 2.48-2.72 ppm; the peaks corresponding to the methyl and benzyl ring protons are observed at 1.93 ppm and 7.40-7.90 ppm, respectively, and the methyl protons of the trimethyl silyl group added to the CPADB compound are found at 0.18 ppm, indicating the successful synthesis of the structure. Based on these results, the TSA-CPADB compound was successfully synthesized.

[0094] In the FTIR spectrum of the CPADB agent (Figure 11), absorption bands due to -COOH stretching vibrations are observed at 3170-2475 cm⁻¹, C=O group (ester carbonyl bond) stretching vibrations at 1745 cm⁻¹, C≡N vibrations at 2230 cm⁻¹, and C=C vibrations at 1425 cm⁻¹ In the TSA-CPADB agent, the bands at 2850-2940 cm-1correspond to both symmetricand asymmetric C-H stretching vibrations, while the bands at 760 cm-1and 840 cm-1are characteristic peaks of the trimethyl silyl group (-Si-(CH3)3). Additionally, in both spectra, the -C=S and C-S bands are observed at 1040 cm-1and 640

[0095]

[0096] cm respectively.

[0097] Synthesis of Monosaccharide Monomer

[0098] The synthesis of l-0-methacryloyl-2,3:4,5-di-0-isopropylidene- / ?-D-fructopyranose (zprF MA)

[0099] In the procedure applied for the synthesis of the iprFr₁MA monomer, first, 4 out of the 5 hydroxyl groups in fructose were protected with isopropylidene groups. Subsequently, the protected fructose was reacted with methacrylic anhydride in the presence of pyridine and DMAP, resulting in the introduction of a methacrylate unit to the fructose compound.

[0100]

[0101] Scheme 5. Synthesis of l-O-methacryloyl-2,3:4,5-di-O-isopropylidene- / ?-D- fructopyranose (z / ?rFr2MA).

[0102] Upon examining the 'H-NMR spectrum of the 2,3:4,5-di-O-isopropylidene- / ?-D-fructopyranose (ip F i) compound (Figure 12), the protons of the methyl groups are observed between 1.56-1.37 ppm, the proton peak of the -OH group at 2.25 ppm, the protons of the -CH2 group labeled (e) in the structure between 3.62-3.78 ppm, and the protons of the fructose ring between 3.79-4.63 ppm, confirming the successful synthesis of the structure.

[0103] When the relevant spectrum given in Figure 13 is examined, the vinyl protons of the methacrylate group in the monomeric structure are clearly observed in the range of 5.61-6.16 ppm, and the peak corresponding to the methyl protons of the methacrylate group is distinctly seen at 1.98 ppm. Thus, it is evident that the iprFr₂MA sugar monomer was successfully synthesized.

[0104] Additionally, the successful synthesis of the iprFr₂MA sugar monomer was confirmed by FTIR measurements. Accordingly, after the ketalization reaction, the broad peak at 2900-3450 cm⁻¹corresponding to the hydroxyl groups of the sugar, disappeared. In the iprFr₂ spectra, characteristic bands of the sugar unit were observed: the -OH stretching band at 3513

[0105]

[0106] cm the -CH stretching bands at 2800-3000 cm⁻¹ and the vibration bands corresponding to the characteristic C-O and C-C groups of carbohydrates in the range of 600-1500 cm

[0107] After the methacrylation reaction, the -OH stretching band, which was distinctly observed at 3500 cm ' in fructose, disappeared, and the intensity of the -CH stretching bands at 2800-3000 cm1increased. Furthermore, bands corresponding to -C=O stretching and -C=C- vibrations were clearly observed at 1650 cm1and 1500 cm respectively (Figure 14).

[0108] Synthesis of N-(3,4-dihydroxyphenethyl)methacrylamide (DopMA) monomer

[0109] The other component in the tetrablock copolymer that carries the magnetic nanoparticles (FesC ) is dopamine methacrylamide monomer. It was obtained as a result of the amidation reaction of 4-(2-aminoethyl)benzene-l,2-diol with methacryloyl chloride in the presence of tri ethylamine. The product was characterized by 'H-NMR analysis. (Figure 15).

[0110]

[0111] Scheme 6. Synthesis of N-(3,4-dihydroxyphenethyl)methacrylamide (DopMA)

[0112] monomer.

[0113] In the FTIR spectrum of the DopMA monomer in Figure 16, the 1645 cm-1absorption peak belonging to the -C=O stretching is clearly seen after the methacrylate unit is added to the compound.

[0114] Synthesis of 2-(4-Formyl-l,3-dioxo- l,3,3a,4,7,7a-hexahydro-2H-4,7-epoxysoindol-2-yl)ethylmethacrylate monomer (MiMA)

[0115] The synthesis of the monomer carrying the furan-protected mal eimide functional group, which has the ability to release drugs, is carried out in four steps. Methacrylate functionality was imparted to the compound by the esterification reaction carried out with methacryloyl chloride in the presence of triethylamine.

[0116] The product formed as a result of protecting the aldehyde group of the furfural compound with ethylene glycol was purified by vacuum distillation method.Etilen glikol

[0117]

[0118] p-TS-OH, benzen

[0119] Scheme 7. Synthesis of 2-(Furan-2-yl)-l,3-dioxolane (FurKet).

[0120] The 'H-NMR analysis of the 2-(furan-2-yl)-l,3-dioxolane (FurKet) compound (Figure 17) proves that the transparent colored liquid substance, which contains peaks for all protons and whose integrations are compatible, has been successfully synthesized.

[0121] When the structure of the FurKetMi (3aS,4R,7R,7aR)-4-(l,3-dioxolan-2-yl)-3a,4,7,7a-tetrahydro-4, 7epoxysobenzofuran-l, 3-dione) compound, which was formed as a result of the Diels-Alder reaction of the FurKet compound with maleic anhydride, was examined by1H-NMR analysis, it was shown that the peaks belonging to all protons were present and the integrations were compatible, proving that the compound was successfully synthesized (Figure 18).

[0122]

[0123] Scheme 8. Synthesis of (3aS,4R,7R,7aR)-4-(l,3-dioxolan-2-yl)-3a,4,7,7a- tetrahydro-4,7 epoxysobenzofuran-1, 3-dione (FurKetMi).

[0124] In the ¹H-NMR analysis (Figure 19) of the (3aS,4R,7R,7aR)-4-(1,3-dioxolan-2-yl)-2-(2-hydroxyethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (FurKetMi OH) compound, which was formed as a result of the reaction of the FurKetMi compound with ethanol amine, the appearance of the methylene peaks at 3.73 ppm and the appearance of the OH peak at 2.37 ppm confirm that the compound was obtained in pure form.

[0125]

[0126] Scheme 9. Synthesis of (3aS,4R,7R,7aR)-4-(l,3-dioxolan-2-yl)-2-(2- hydroxyethyl)- 3a,4,7,7a-tetrahydro-lH-4,7-epoxysoindole-l,3(2H)-dione

[0127] (FurKetMiOH).As the last step, 2-((3aS,4R,7R,7aR)-4-(l,3-dioxolan-2-yl)-l,3-dioxo-l,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl methacrylate monomer (MiMA) was synthesized by reacting FurKetMiOH with methacryloyl chloride.

[0128]

[0129] Scheme 10. Synthesis of 2-((3aS,4R,7R,7aR)-4- (l,3-Dioxolan-2-yl)-l,3-dioxo- l,3,3a,4,7,7a-hexahydro-2H-4,7-epoxysoindol-2-yl) ethyl methacrylate (MiMA).

[0130] MiMA monomer was isolated by column chromatography (silica gel 60 A, 70-230 mesh) using ethyl acetate: hexane (1:2) eluent system. The solvent mixture was removed under vacuum and a dark brown viscous product was obtained. The substance was dissolved with ethyl acetate and precipitated in hexane. It was kept at -18 °C overnight. A white solid product was obtained. The chemical structure of the obtained monomer was confirmed by 'H-NMR analysis (Figure 20). Functional group analyses of MiMA monomer and precursor compounds were also confirmed by FTIR (Figure 21).

[0131] Synthesis of Lysine monomer (fBoc-Lys-NCA)

[0132] Lysine monomer, which will undertake the siRNA transport task, was synthesized with a t-Boc-protecting group using the following method. In this direction, in the synthesis of lysine monomer, firstly the amino groups were protected with di-te / 7-butyl dicarbonate compound and then N-( / c77-butoxy carbonyl)-L-lysine N-carboxyanhydride ( / Boc-Lyz-NCA) monomer was obtained by ring closure reaction. The chemical structure of the obtained compound was confirmed by 'H-NMR analysis (Figure 22).

[0133] o

[0134] Cl ' A OCCI3

[0135] TMSCI, Et3N

[0136]

[0137] Scheme 11. Synthesis of N-( / c / 7-butoxycarbonyl)-L-lysine N-carboxyanhydride

[0138] compound.In Figure 23, the structures were elucidated by taking FTIR spectra for each step of the ZBoc-Lys-NCA synthesis. When the relevant spectrum is examined, there is a distinct C=O band at 1690 cm-1, N-H bending at 1630 cm-1and specific bands belonging to C-O stretching at 1164 cm-1when the spectrum of ZBoc-Lys is examined. When the FTIR spectrum of the ZBoc-Lys-NCA compound is examined, N-H stretching at 3370 cm-1and C=O stretching coming from N-carboxyanhydride at 1850 cm-1and 1790 cm-1are observed in the spectrum, indicating that the structure was synthesized successfully.

[0139] Polymerization Reactions

[0140] After the successful synthesis of all organic compounds, preparation of polymers was started. In this context, polylysine polymer was prepared by ring-opening polymerization method and triblock glycopolymer was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization technique.

[0141] Synthesis of co-azido functional N-ZerZ-butyloxycarbonyl-L-lysine polymer (P(ZBoc-Lys)-N3))

[0142] Synthesis of P(ZBoc-Lys)-N3 peptide polymer by ring-opening polymerization was carried out at 0 °C with the ratio [M]: [I] =

[0050] :

[0001] and monomer as 1 g. The polymer was dissolved with THF and precipitated in diethyl ether to remove unreacted monomer.

[0143]

[0144] Scheme 12. Synthesis of co-azido-functional N-ZerZ-butyloxycarbonyl-L-lysine polymer.

[0145] Ring-opening polymerization was repeated twice and the polymerization conditions and molecular weight characterizations of the obtained polymers are summarized in Table 1. A monomodal peak was obtained in THF-GPC analysis.Table 1. Molecular weight characterizations of Pf / Boc-LysJ-Ns homopolymers.

[0146] No Polymer Name [MJ: [I] f / o) A / H, GPC D

[0147]

[0148] 1 P( / Boc-Lys)-N3 50:1 4 28 3300 2650 1.36

[0149] 2 P( / Boc-Lys)-N3 50:1 4.5 30 3500 4450 1.22

[0150] In addition, the molecular weight and end group analysis of the Pf / Boc-LysJ-Ns polymer were calculated as 28% and 30%, respectively, by NMR analysis. (Figure 24).

[0151] Poly(2-(4-(l,3-dioxolan-2-yl)-l,3-dioxo-l,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl methacrylate) homopolymerization (PMiMA)

[0152] Polymerization was carried out at 70 °C with the ratio [M]: [RAFT]:[I]=40: 1:0.125 and the monomer was 2 g. The polymer was dissolved with acetonitrile and precipitated in diethyl ether to remove unreacted monomer.

[0153] RAFT polimerizasyonu

[0154]

[0155] Scheme 13. PMiMA homopolymer synthesis.

[0156] The molecular weight of the homopolymer obtained was determined by ¹H-NMR analysis (Figure 25B) and THF-GPC (Figure 25 A). Polymerization conditions and results are summarized in Table 2. Monomodal Gaussian peak with a molecular weight of 2650 and a polydispersity index of 1.25 was obtained from THF-GPC calibrated according to polymethylmethacrylate standards. In addition, the molecular weight and end group analysis were performed by NMR analysis and the monomer conversion was calculated as 26%. The molecular weight calculated from NMR was found to be Mn= 4250 g / moL. As a result of ¹H-NMR analysis, the repeating unit (DPn) of the polymer was found to be 11. In order to increase the amount of drug to be loaded, the polymerization time was extended in the second polymerization and the repeating unit of the polymer was increased and calculated as 14 from NMR.Table 2. Molecular weight characterizations of PMiMA homopolymer.

[0157] .. Polymer....... Time Con.,,,,,,

[0158] No.TJ[MJ: [RAFT]: [I].... A / n.vm Afn.teo V / n.(,i’( D Name (hours) (%)

[0159] 1 PMiMA 40:1:0.125 7 26 4250 4050 2650 1.25 2 PMiMA 40:1:0.125 9 35 5300 5900 2860 1.35

[0160] Poly(2-(4-(l,3-dioxolan-2-yl)-l,3-dioxo-l,3,3a,4,7,7a-hexahydro-2H-4,7-epoxysoindol-2- yl)ethyl methacrylate- / > / oc£-N-(3,4-dihydroxyphenethyl)methacrylamide) diblock polymerization P(MiMA-ft-DopMA)

[0161] Diblock polymerization was carried out at 70 °C with the ratio [M]:[MacroRAFT]:[I]=33:l:0.135 and homopolymer was 0.5 g and DopMA monomer was 1.43 g. The polymer was dissolved with acetonitrile and precipitated in diethyl ether to remove unreacted monomer.

[0162]

[0163] Scheme 14. Synthesis of P(MiMA-Z>-DopMA) diblock polymer.

[0164] The molecular weight of the obtained diblock copolymer was elucidated by THF-GPC and ¹H-NMR analysis (Table 3). It was determined by ¹H-NMR analysis that 3 units of monomer were polymerized (Figure 26). When the polymerization ratio of the monomer: Macro-RAFT agent was kept at 40, the monomer conversion was calculated as 10% in the same period. The molecular weight characterizations of the prepared P(MiMA-Z>-DopMA) diblock copolymer are summarized in Table 3.

[0165] Table 3. Molecular weight characterizations of P(MiMA-b-DopMA) diblock copolymer... Polymer nxi. iirT1Time Con. A / H, NM,, A / n, GP

[0166] No. [M]: [RAFT]: [I] Mn,teo D Name (hours) (%) R C

[0167] 1 P(MiMA- 33:1:0.135 8,5 9 4900 8300 3450 1.34

[0168] 6-DopMA) 2 P(MiMA- 40:1:0.135 8,5 10 5150 7600 2680 1.36

[0169]

[0170] 6-DopMA)Poly(2-(4-(l,3-dioxolan-2-yl)-l,3-dioxo-l,3,3a,4,7,7a-hexahydro-2H-4,7-epoxysoindol-2-yl)ethylmethacrylate- / > / oc -N-(3,4-dihydroxyphenethyl)methacrylamide)- / > / oc -l-0-methacryloyl-2,3:4,5-di-0-isopropylidene- / ?-D-fructopyranose) triblock copolymerization (P(MiMA- / z-l)op. MA- / z-z / ?rFr2. MA))

[0171] Triblock polymerization reaction was carried out at 70 °C by weighing 0.20 g of diblock copolymer (P(MiMA-Z>-DopMA) and 2.529 g of z / vFnMA monomer in the ratio [M]:[MakroRAFT]:[I]=200:l:0.175. The polymer was dissolved with acetonitrile and precipitated in diethyl ether to remove unreacted monomer.

[0172]

[0173] Scheme 15. Synthesis of P(MiMA-Z>-DopMA-Z>-z / ?rFr2MA) triblock polymer.

[0174] The chemical structure of the obtained product was elucidated by ¹H-NMR analysis and THF-GPC (Figure 27). ¹H-NMR analysis determined that 92 units of sugar monomer were polymerized.

[0175] Table 4. Molecular weight characterizations of P(MiMA-Z>-DopMA-Z>-zprFr2MA) triblock copolymer.

[0176] No Polymer [MJ: [RAFT]: [I].im\C°n‘ / „. SMR A / „.teo A / „.(,i>( D Name (hours) (%)

[0177] 1 P(MiMA-Z>- DopMA-Z>- 200:1:0.175 7.5 46 35350 39000 42300 1.38 z / vFnMA)

[0178] (Hydrolysis of trimethylsilyl group of P(MiMA- / >-DopMA- / >-zprFr2MA) triblock copolymer)

[0179] P(MiMA-Z>-DopMA-Z>-z / ?rFr2MA) triblock copolymer was dissolved with THF and the reaction was carried out at room temperature in the presence of TBAF (0.025 mL, 1 M) solution.

[0180]

[0181] Scheme 16. TBAF hydrolysis reaction of P(MiMA-Z>-DopMA-Z>-zprFr2MA)

[0182] triblock polymer.

[0183] According to ¹H-NMR analysis, it was determined that trimethyl silyl groups, which appeared at 0.19 ppm with the hydrolysis reaction, disappeared. The TBAF hydrolysis reaction efficiency was calculated as 100% from NMR (Figure 28).

[0184] Synthesis of P(MiMA- / >-DopMA-b-zprFr2MA)- / >-P(fBoc-Lys) tetrablock copolymer via click reaction

[0185] Copper-catalyzed azide-alkyne cycloaddition reaction between the prepared triblock copolymer (P(MiMA-A-DopMA-A- / / 9 / 'Fr2MA) and polylysine homopolymer (P( / Boc-Lys)-N3) was carried out in high yield.

[0186]

[0187] Scheme 17. Synthesis of P(MiMA-Z>-DopMA-Z>-z / ?rFr2MA)-Z>-P(tBoc-Lys)

[0188] tetrablock copolymer via click reaction.

[0189] The molecular weights of the tetrablock copolymers obtained after the click reaction, determined from DMF-GPC and NMR, are given in Table 5. The chemical structure of the obtained product was elucidated by DMF-GPC and ¹H-NMR analysis (Figure 29). The presence of the proton belonging to the triazole group and the protons belonging to the P(tBoc-Lys)polymer at 8.03 ppm in the NMR spectrum shows that the click reaction has been successfully performed. It is clearly seen in the GPC chromatogram that the tetrablock copolymer with a monomodal gaussian peak and narrow molecular weight distribution was obtained.

[0190] Table 5. Molecular weight characterizations of tetrablock copolymer.

[0191] No Polymer Name MI, NMR V / n.teo A / n.c.pc 1) 1 P(MiMA-b-DopMA-b-iprFr₁MA)-b-P(tBoc- 43250 44000 48450 1.26 Lys) 2 P(MiMA-b-DopMA-b-iprFr₂MA)-b-P(tBoc- 38650 39400 42500 1.28 Lys)

[0192]

[0193] Hydrolysis of isopropylidene and tertbutyloxycarbonyl group of P(MiMA- / >-DopMA- / >- iprFr2MA)- / >-P(fBoc-Lys) tetrablock copolymer

[0194] The isopropylidene groups carried by the sugar segment in the tetrablock copolymer, the ketal groups from the MiMA monomer, and the / c / 7-butoxy groups carried by the lysine polymer were removed in a single step by acidic hydrolysis in the presence of trifluoroacetic acid (TFA).

[0195]

[0196] Scheme 18. TFA hydrolysis of P(MiMA-Z>-DopMA-Z>-z / ?rFr2MA)-Z>-P(tBoc-Lys)

[0197] tetrablock copolymer.

[0198] The chemical structure of the obtained P(MiMA-Z>-DopMA-Z>-Fr2MA)-Z>-P(Lys) tetrablock copolymer was elucidated by DMF-GPC and 'H-NMR analysis (Figure 30). The exact name of the obtained P(MiMA-Z>-DopMA-Z>-FrMA)-Z>-P(Lys) copolymer is Poly(2-(4-(l,3-dioxolan-2- yl)-l,3-dioxo-l,3,3a,4,7,7a-hexahydro-2H-4,7-epoxysoindol-2-yl)ethylmethacrylate-Z> / oc -N- (3,4-dihydroxyphenethyl)methacrylamide)-Z> / oc -l-O-methacryloyl-2,3:4,5-di-O- isopropylidene- ^D-fructopyranose)-Z> / ocA Poly(L-lysine) and the determined molecular weights of the tetrablock copolymer are summarized in Table 6.Table 6. Molecular weight characterizations of P(MiMA-Z>-DopMA-Z>-Fr2MA)-Z>-P(Lys) polymer.

[0199] No Polymer Name MI, NMR V / n.teo V / n.(,i>( 1) 1

[0200] P(MiMA-Z>-DopMA-Z>-Fr2MA)-Z>-P(Lys) 29200 29950 32100 1.36

[0201] Doxorubicin loading onto P(MiMA- / >-DopMA- / >-Fr2MA)- / >-P(Lyz) tetrablock copolymer (P-Dox)

[0202] The active ingredient doxorubicin was chemically incorporated into the tetrablock copolymer via imine bond.

[0203]

[0204] Scheme 19. Doxorubicin loading into P(MiMA-Z>-DopMA-Z>-Fr2MA)-Z>-P(Lys)

[0205] tetrablock copolymer.

[0206] The complete disappearance of the peak belonging to the -CHO proton at 9 ppm and the emergence of peaks belonging to Dox in Figure 31 proves that all aldehyde groups are saturated with Dox. In addition, the determined molecular weights of the drug-loaded tetrablock copolymer are summarized in Table 7.

[0207] Table 7. Molecular weight characterizations of P(MiMA-A-DopMA-A-Fr2MA)-A-P(Lys) / Dox polymer.

[0208] No Polymer Name MI, NMR V / n.teo MI, GPC 1) 1 P(MiMA-Z>-DopMA-Z>-Fr2MA)-Z>-P(Lys) / Dox 37300 38050 39000 1.32

[0209] Synthesis of Magnetic Nanoparticles (FesC )

[0210] Since large nanoparticles (Figure 1) were obtained as a result of DLS measurements taken in the first method applied in the synthesis of FesC nanoparticles as a contrast enhancer and photothermal therapy agent in MRI, a different procedure applied at higher temperatures was tried to obtain smaller nanoparticles.The procedure was carried out as follows; iron(III) chloride hexahydrate and sodium oleate were dissolved in a mixture of ethanol, deionized water, and hexane. The mixture was stirred for half an hour under nitrogen at room temperature and then the temperature was increased to 70 °C. The reaction was stirred for 4 hours and then brought to room temperature. Extraction was performed 3 times with deionized water and a dark brown liquid material was obtained. Then, the iron-oleate complex, oleic acid, and 1 -octadecene were mixed at room temperature until they became a homogeneous solution and the temperature was slowly increased to 300 °C. After stirring at this temperature for one hour, the reaction was brought to room temperature. Ethanol was added to the mixture and the nanocrystals formed were separated by centrifugation. Then, they were dispersed with chloroform and their structures were elucidated by DLS and HR-TEM. As a result, it was determined that nanocrystals of approximately 4 nm size were obtained (Figure 2).

[0211] In the last method, magnetic nanoparticles that can be dispersed in water were synthesized using a co-precipitation method containing Fe+2and Fe+3ions in a 1:2 molar ratio. Then, they were dispersed with water and DLS and TEM analyses were performed (Figure 3).

[0212] Preparation of Upconversion Nanoparticles (UCNP)

[0213] UCNP with appropriate shape and size, which forms the core of the nanotheranostic bionanoprobe and gives emission when excited with a 980 nm NIR laser, was synthesized.

[0214] Preparation of NaYF4: Yb3+ / Er3+UCNP (UCNP)

[0215] NaYF4: Yb3+ / Er3+(UCNP) nanocrystals consisting of NaYF4doped with 20% Yb3+and 2% Er3+were fabricated by the solvothermal method at high temperatures according to the procedure reported by Liu et al.. TEM and DLS results confirmed that UCNPs were successfully synthesized (Figure 4). These results indicate that the synthesized UCNPs have a uniform structure with a size of ~35 nm in hexane.

[0216] Also shown in Fig. 6 is the TEM / EDX spectrum analysis of NaYF4: Yb3+ / Er3+. Prominent peaks representing F, Na and Y atoms within the nanoparticle are shown at 0.62, 0.94 and 2.05 keV, respectively. Peaks recorded at 0.22 and 8.04 keV correspond to the presence of C and Cu atoms on standard supporting TEM grids. Weaker peaks observed at 1.48, 7.40 and 8.40 keV are attributed to Yb atoms, while the signal from Er atoms (~6.9 keV) could not be detected due to their low concentration.Synthesis of citrate-coated UCNP (UCNP-C)

[0217] To transfer UCNP from hexane to water, ligand exchange was performed by stirring in 0.1 M trisodium citrate buffer solution and citrate-coated UCNPs were synthesized. The particle size of citrate-coated nanocrystals was elucidated by DLS and TEM. As shown in Figure 5, UCNP-C was synthesized with a uniform distribution of ~40 nm in size.

[0218] Preparation of Tetrablock Polymer Coated UCNP Nanoprobes (UCNP@P-Dox)

[0219] UCNP nanoparticles were coated with tetrablock polymer to prepare biocompatible theranostic nanoplatform. For this, NaYF4: Yb3+ZEr3+nanocrystal and P(MiMA-Z>-DopMA-Z>-Fr2MA)-Z>-P(Lys) / Dox (P / Dox) polymer were mixed at room temperature. The obtained UCNP@P-Dox nanoprobe was illuminated by DLS, FTIR and TEM analysis (Figure 33). According to TEM and DLS results, the size of UCNP@P-Dox was determined as -135 nm.

[0220] Preparation of Magnetic Glycopeptide Bionanoprobes (UCNP@MP-Dox)

[0221] The magnetic FesCh nanoparticles were attached to the catechol (DopMA) groups in the tetrablock copolymers to impart magnetic properties to the tetrablock copolymer. The obtained UCNP@MP-Dox nanoprobe was elucidated by DLS and TEM analysis (Figure 33). After the modification of UCNP@P-Dox with Fe3O4 nanoparticles, its size was determined as -190 nm. In addition, the detailed analysis of the FTIR spectra of UCNP, UCNP-C, Fe3O4, P-Dox, UCNP@P-Dox and UCNP@MP-Dox is shown in Figure 33B. The characteristic -OH and C-H stretching vibrations observed at ~ 3550-3200 cm'1and 3000-2840 cm'1, respectively, confirm the presence of polymer on the surface of the nanoparticles. The spectrum of UCNP-C exhibits an absorption band at 1596 and 1410 cm'1due to the C=O stretching vibrations of citric acid, while the spectrum of Fe3O4 exhibits absorption bands at 3433, 1620 and 570 cm'1attributed to the -OH, C=O, and Fe-0 stretching vibrations. After coating UCNP-C with P-Dox, the stretching vibrations of the C=O groups of P-Dox at 1735 cm'1are observed in the spectrum. A similar peak is observed in the spectrum of UCNP@MP-Dox at 570 cm'1; This proves that UCNP@P-Dox is coated with magnetic nanoparticles.

[0222] The ^-potential values of UCNP-C, Fe3O4, P-Dox, UCNP@P-Dox, UCNP@P-Dox / siRNA, UCNP@MP-Dox, and UCNP@MP-Dox / siRNA nanoprobes are shown in Figure 7A. Before coating with P-Dox, the ^-potential of UCNP-C was approximately -23.00 ± 0.54 mV due tothe coating of UCNP surface with citrate. The ^-potential of Fe3O4 and P-Dox was determined as -17.75 ± 1.06 mV and +18.95 ± 0.86 mV, respectively. After coating of UCNP-C with cationic glycopeptide polymer, the ^-potential was measured as +13.90 ± 0.80 mV. It is then shown that when FesCf nanoparticles are incorporated onto the UCNP@P-Dox surface, the zeta potential value decreases to +8.20 ± 0.44 mV.

[0223] The upconversion photoluminescence (UCL) properties of magnetic nanoparticles loaded with UCNP, UCNP-C, UCNP@P-Dox and UCNP@MP-Dox (1 mg / mL) were determined under NIR excitation at 980 nm wavelength (Fig. 7B). The typical upconversion emission of Er3+was found to originate from the transitions at 410 nm (2H9 / 2 —>4Irs / 2), 525 nm (2Hn / 2 —>4IIS / 2), 542 nm (4S3 / 2 —>4Ii5 / 2) and 656 nm (4Fg / 2 —>4Irs / 2). It is observed that the emission intensity slightly decreased as a result of citrate coating carried out to transfer UCNP from hexane to water. Another analysis, thermogravimetric analysis (TGA), provides evidence that UCNPs were effectively surface modified with P-Dox and FesCf (Figure 7C). Over the entire temperature range, the weight loss observed for UCNPs was approximately 9.4% due to the OA coating. UCNP-C started to degrade at approximately 200 °C, and a significant mass loss of 25% was observed between 200 and 400 °C. While the weight loss of Fe3O4 nanoparticles was only 5.3%, the weight loss of P-Dox was found to be 99.2% in the same temperature range. In addition, the weight loss of UCNP coated with P-Dox was approximately 54.1%, while this loss was reduced to 30.1% with the addition of FesCU nanoparticles to the structure. According to TGA data, the amount of P-Dox and FesCU coating on the UCNP surface is about 27.3% and 24%, respectively.

[0224] In addition, X-ray diffraction (XRD) analysis for NaYF4: Yb3+, Er3+, FesCU and UCNP@MP-Dox is presented separately in Fig. 7D. It was revealed that the peaks observed in the XRD results of hexagonal phase NaYF4: Yb3+, Er+3matched with the peaks of standard X-ray diffraction JCPDS 28-1192. The sharp peaks in the XRD spectrum are due to the crystal structure of the synthesized UCNP. Similarly, the XRD result of FesCU nanoparticles is also in agreement with those reported previously. The glycopolymer and FesCU coating on the UCNP surface caused the sharpness of the peaks observed in the UCNP@MP-Dox peaks to decrease.Investigation of Magnetic Properties and Photothermal Therapy Efficacy of UCNP@MP-Dox Nanoprobe

[0225] FesCU nanoparticles are widely investigated as photothermal therapy (PTT) agents due to their superior biocompatibility, low toxicity, superparamagnetism, chemical inertness and excellent stability in physiological environment. Using UV-vis spectrophotometry, it was determined that UCNP@MP-Dox (200 pg / mL) showed significant absorption at 980 nm (Fig. 34E). This absorption is an important indicator of NIR (near-infrared) induced photothermal effect and is expected to effectively destroy cancer cells via photothermal therapy with slight temperature increase (41.8 °C - 45 °C) at minimum laser intensity (980 nm). According to the standards set by the American National Standards Institute for the safe use of lasers, the maximum permissible exposure (MPE) value for a 980 nm laser is 0.72 W / cm2

[0226] In order to evaluate the photothermal properties of UCNP@MP-Dox nanoprobes, 980 nm laser irradiation was performed for 720 s at different power levels (0.50, 0.60, 0.72, 0.85, and 1.00 W / cm2). The temperature change in the solution was monitored by an IR thermal camera. The results show that the temperature increase increases as the nanoparticle concentration and laser intensity increase. Compared to the initial temperature of UCNP@MP-Dox solution (200 pg / mL), a temperature increase of 17.7 °C with 0.50 W / cm2and 31.6 °C with 1.00 W / cm2is observed after 720 s of laser irradiation (Figure 34A). As expected, the temperature increase increased in direct proportion to the irradiation time and laser power density. When the laser power was changed to 0.85, 0.72, and 0.60 W / cm2, the temperature increases were recorded as 27.80, 25.05, and 20.75 °C, respectively. In the irradiation with a power of 0.72 W / cm2at 980 nm, it was determined that the temperature increase decreased with the decrease in solution concentrations. According to the data, the temperature increases at concentrations of 400, 200, 100, 75, 50 and 25 pg / mL were measured as 28, 25.05, 22.20, 19.60, 18.50 and 15.20 °C, respectively (Figure 34B). It is shown that UCNP@MP-Dox, when irradiated at a wavelength of 980 nm and a laser intensity of 0.72 W / cm2, provides sufficient temperature increase to effectively destroy tumor cells, thus being a suitable photothermal therapy agent.

[0227] Furthermore, the photothermal conversion efficiency of UCNP@MP-Dox was evaluated by comparing its components (UCNP-C, FesCU, and P-Dox) against the deionized water control group (Fig. 34C). Under the same conditions, only a slight increase of 4.8 °C was observed in the temperature of water, while this increase was recorded as 25 °C in UCNP@MP-Dox.Photothermal conversion efficiency ( / ) is an important parameter that indicates the effectiveness of photothermal treatment. For this purpose, the photothermal conversion efficiency of UCNP@MP-Dox was calculated by a previously reported method. The photothermal conversion efficiency ( / ) of UCNP@MP-Dox nanoparticles was determined using the following equations.

[0228] η = hS(TMax— TSurr) - QDis

[0229]

[0230] I(1 − 10−A₉₈₀)

[0231] The hS value in equation 1 is obtained through equation 4 and Figure 34D. Here h represents the heat transfer coefficient and S represents the surface area of the container. In order to calculate the hS value, the 0 value was calculated from equation 2 based on the data obtained from the natural cooling graph in Figure 34D and the negative slope line between ln(0) and t was drawn (Figure 34D). The time constant of heat transfer (rs) was calculated as 279 s. In addition, CD (4.2 J / °C) represents the heat capacity and mo (0.3 g) represents the mass of water in equation 4. Therefore, according to this equation, the hS value is 4.5 mW / °C.

[0232] θ = (T − TSurr) / (TMax− TSurr) (2)

[0233] t = -TsIn(0) (3)

[0234]

[0235] The UCNP@MP-Dox solution reached the maximum constant temperature (TMax) of 51.6 °C, while the ambient temperature (TSurr) remained at 26.5 °C. Therefore, the temperature difference (TMax-TSurr) in the UCNP@MP-Dox solution is 25.1 °C. The laser power (I) is 0.72 W, and the absorbance of UCNP@MP-Dox at 980 nm (η) is 0.38 (Fig. 34E). QDisrepresents the heat released due to the absorption of light by the solvent and the container. From these calculations, the photothermal conversion efficiency (η) was found to be 27% when the UCNP@MP-Dox nanoparticle was excited by the 980 nm laser.

[0236] Figure 34F shows that the photothermal performance of UCNP@MP-Dox nanoprobes (200 pg / mL) was unchanged after five cycles of 12 min irradiation (laser on) followed by 12 min cooling (laser off) at a power density of 0.72 W / cm2, proving the remarkable photothermal stability of UCNP@MP-Dox nanoprobes.The magnetic properties of the prepared UCNP@MP-Dox and Fe3O4 nanoparticles were investigated by measurements at room temperature using a vibrating sample magnetometer (VSM). The saturation magnetization (Ms) values were determined as 84.65 emu.g1for FesCh nanoparticles and 33.00 emu.g1for UCNP@MP-Dox nanoprobe (Figure 35 A). This significant decrease in the Ms value of UCNP@MP-Dox can be attributed to the non-magnetic polymer shell surrounding FesC. However, it is seen that UCNP@MP-Dox can be quickly and easily separated from an aqueous solution using an external magnetic field.

[0237] In addition, as seen in Figure 35B, T2-weighted MR imaging was performed with seven different dilutions of UCNP@MP-Dox, and a gradual decrease in signal is observed with increasing Fe concentration (3T, TR = 3000 ms, TE = 60 ms). The relaxation coefficient (n value) obtained from the slope of this graph was calculated as 30.138 s-1mM-1(Figure 35B). As a result, UCNP@MP-Dox produces remarkable contrast in T2-weighted imaging and offers a high potential as an MRI agent that can guide treatment thanks to this feature.

[0238] Dox Release Experiment of Prepared Nanoprobes

[0239] The imine bond, which can be broken down in acidic media but is stable under normal physiological conditions, was used as a linker between the glycopeptide polymer and Dox, thus providing pH-sensitive Dox release. The drug release profiles of P-Dox, UCNP@P-Dox and UCNP@MP-Dox were examined using a UV-Vis spectrophotometer at 37 °C in different pH environments (pH 5.5, 6.8 and 7.4). These pH values reflect the acidic environment of the tumor microenvironment and the neutral pH of healthy tissues. The drug release profiles of P-Dox (Figure 36A), UCNP@P-Dox (Figure 36B) and UCNP@MP-Dox (Figure 36C) nanoprobes exhibit similar properties. Dox release from UCNP@MP-Dox was measured as 39.5% at pH 7.4 and 74% at pH 5.5 after 96 h. As expected, the pH-sensitive release profile shows that Dox is rapidly released as a result of the cleavage of the imine bond in acidic media. These results suggest that UCNP@P-Dox and UCNP@MP-Dox release more Dox in the tumor microenvironment compared to healthy tissues.

[0240] As shown in Figure 36D, the Dox release profile was examined by analyzing the increasing absorbance at 488 nm at certain time intervals under laser irradiation in different pH media. Dox release showed a sharp increase during 15 min of laser irradiation, while this release decreased when no irradiation was applied. As a result, Dox release reached 37.5% at the end of 4 hours under laser irradiation at pH 5.5, while this rate was determined as 15.5% when laserirradiation was not applied (Figure 36D). Similarly, Dox release at the end of 4 hours under laser irradiation at pH 6.8 and pH 7.4 was 21.5% and 14.5%, respectively, while these values were measured as 13% and 7.5% when no irradiation was applied, respectively. It should be noted that the cleavage of the imine bond was significantly affected by increasing [H+] concentration and temperature. At higher pH and temperature, the cleavage of the imine bond increased, leading to improved drug release. These results show that NIR light-induced hyperthermia increased the Dox release from UCNP@MP-Dox.

[0241] Preparation of UCNP@P-Dox Carrying siRNA and Investigation of Their Release Profiles (UCNP@P-Dox / siRNA)

[0242] For loading siRNA, stock solution of Bcl-2 siRNA was prepared in ultrapure water (nuclease-free distilled water) [20nM], In order to detect the interaction between siRNA and nanoparticles, complexes were formed by utilizing the negative charge of siRNA and the positive charge of polymer-coated bionanoprobes and prepared at 7 different NP / siRNA (0.25, 0.5, 1, 1.5, 2, 4, 5) ratios. Complex formation with Bcl-2 siRNA was examined using the agarose gel running method. In this experiment, the position of uncomplexed Bcl-2 siRNA bands was monitored by staining with EtBr and using its strong fluorescence property. Low-temperature 2.5% agarose gel was prepared with IX TAE (Tris-Acetate-EDTA). Sample was loaded with 6X DNA loading buffer in a final volume of 2 pL per well. It was run for 45 minutes at 100V with IX TAE containing 0.1 mg / mL EtBr and the gel image was taken with the Biorad Chemi Doc imaging system. The results of the preparation of UCNP@P-Dox carrying siRNA and the evaluation of binding rates in agarose gel electrophoresis are given in Figure 37. The experiments were continued by selecting the NP / siRNA ratio value of 5, at which the amount of free siRNA was not observed.

[0243] The sizes, charges, polydispersity indexes, and zeta potentials of the prepared UCNP@P-Dox and siRNA-loaded nanoparticles were measured by DLS, and some results are summarized in Table 8. As expected, the ^-potential value of UCNP@P-Dox changed significantly after loading with anti-Bcl-2 siRNA, confirming that siRNA was effectively loaded into UCNP@P-Dox.Table 8. Size and zeta potential values of bionanoprobes at specific nitrogen / phosphorus (N / P) ratios.

[0244] No UCNP@MP-Dox siRNA N / P Boyut (nm) PDI (mV) 1 - 1 - 658 0.45 -16.71 2 0.25 1 0.25 686 0.73 -6.57 3 0.5 1 0.5 640 0.94 -4.72 5 5 1 5 83 0.52 -0.12 6 1 - 1 102 0.24 2.69

[0245] Release studies of siRNA were carried out in a shaking orbital shaker at 37 °C. An amount of the prepared siRNA-loaded nanoparticle solution was taken onto a semipermeable dialysis membrane (MWCO 100 kDa, 2 mL) and the ends were closed with standard clips. The structural integrity of siRNA in samples taken at certain time intervals was determined by gel electrophoresis (Figure 39).

[0246] Testing of Prepared Theranostic Bionanoprobes with In Vitro Studies

[0247] In vitro studies were performed on MCF-7 (estrogen sensitive) and 3T3 / NIH (ATCC® CRL-1658) cell lines. In this context, cytotoxicity studies, intracellular uptake studies and apoptosis studies were performed.

[0248] Cytotoxicity Studies

[0249] For cytotoxicity studies, cells were cultured as planned and tested for 24 hours. The cytotoxicity steps completed for the relevant groups were studied in triplicate for each experiment and repeated as two separate independent experiments. Variance calculations of the obtained absorbance data The IC50 values obtained from two independent experiments were evaluated using the calculation model in equation 6 and summarized for two cell types in Table 9 by taking the average of the IC50 values obtained from two independent experiments. The change in cell viability due to cytotoxicity is presented in Figure 40 for MCF7 and 3T3 cells, respectively.

[0250]

[0251]

[0252] When evaluated in terms of cell viability, it is observed that UCNP and P (polymer) show low toxicity in terms of IC50 and viability percentages in both cell types (minimum IC50 164.08 pg / mL - maximum IC5023.605 pg / mL). For each cell type, in terms of their own IC50 values and viability percentages; Although P- Dox and UCNP@P- Dox groups contain Dox, their cytotoxic effects are lower when Dox is considered (Table 9).

[0253] Table 9. Average IC50 values obtained from two independent experiments.

[0254] IC50 (pg / mL) MCF-7cells 3T3 / NIH cells UCNP 50,94 46,25

[0255] P 37,22 40,07

[0256] P-Dox 4,18 32,43 UCNP@P-Dox 18,87 33,64

[0257] Dox 0,94 0,92

[0258] For all groups, cell viability assessments at different concentrations with and without NIR irradiation (λ = 980 nm, 0.72 W / cm2) were performed using the MTT assay at two different time periods (24 h and 48 h) (Figure 41 and Figure 42). The cytotoxicity results of free Dox and other nanoprobes (UCNP, P and P-Dox) are presented in Figure 40.

[0259] Due to the significant differences between the two cell lines, four concentrations (1.95, 3.90, 7.80 and 15.60 pg / mL) were selected as the most suitable concentrations after 24 h of treatment. It was observed that the cytotoxic effects were reduced compared to the higher doses and the untreated group. Especially, the control group, 3T3 cells, showed significantly higher viability percentage compared to the Dox group. However, Dox significantly reduced cell viability compared to untreated cells at all concentrations, and this was also observed in MCF-7 cells (Figure 40).

[0260] In MCF-7 cells, treatment with UCNP@P-Dox / siRNA without laser irradiation significantly decreased cell viability compared to the untreated group within 24 h at all concentrations (p<0.0001). Similarly, significant cytotoxicity was observed when MCF-7 cells were incubated with UCNP@MP-Dox / siRNA without laser irradiation (Figure 41). Furthermore, under NIR irradiation, UCNP@MP-Dox and UCNP@MP-Dox / siRNA nanoprobes caused a significantdecrease in the viability of MCF-7 cells compared to the untreated group (p<0.0001; 3.90 pg / mL). On the other hand, a significant decrease in viability was noticed between P-Dox / siRNA and UCNP@P-Dox / siRNA groups at concentrations of 3.90, 7.80 and 15.60 pg / mL. These concentrations were chosen to evaluate intracellular uptake in both cell lines. Figure 42 shows the comparative effects of treatments applied at different concentrations on cell viability after 48 h incubation, both with NIR irradiation (λ = 980 nm, 0.72 W / cm2) and without irradiation. The obtained results showed that UCNP@P-Dox, UCNP@P -Dox / siRNA, UCNP@MP-Dox and UCNP@MP-Dox / siRNA nanoprobes were consistent with the 24 h incubation data.

[0261] Intracellular uptake studies

[0262] Intracellular uptake in each cell group was evaluated for 6th, 12th and 24th hours. The time dependent change of intracellular uptake was observed by evaluating the same doses for two cell types. The selected doses were 3.9, 7.8 and 15.60 pg / mL.

[0263] Flow cytometry measurements were performed after excitation of 3T3 and MCF-7 cells at 488 nm wavelength to perform intracellular uptake analysis using FL1-A channel (Figure 46). Intracellular uptake analysis by flow cytometry in 3T3 cells reveals that UCNP and UCNP@P-Dox exhibit similar rates (fold change below 1) (Figure 46). No significant cellular internalization is observed compared with the untreated group (Figures 43 and 44). On the other hand, Dox treatment shows an increasing uptake rate over time in 3T3 cells, which appears to be associated with decreased cell viability.

[0264] In MCF-7 cells, the intracellular uptake of UCNP@P-Dox was found to be higher than that of the untreated group (unt) at all concentrations (Figures 43 and 44). Particularly, the highest fold change was determined in MCF-7 cells after 24 h of treatment at a concentration of 7.8 pg / mL. Besides, the uptake rate of UCNP does not show any significant change as in 3T3 cells.

[0265] In MCF-7 cells, the intracellular uptake of UCNP@P-Dox at a concentration of 3.9 pg / mL is increased by 2.66, 1.76, and 3.05 folds compared to Dox at 6, 12, and 24 hours, respectively. This increase is observed as 2.07, 1.12, and 3.79 folds at a concentration of 7.8 pg / mL; and 1.96, 1.60, and 3.50 folds at a concentration of 15.60 pg / mL, respectively. In contrast, Dox treatment shows a concentration-dependent increase in uptake depending on the time intervals (Figure 46).After this step, the intracellular uptake of UCNP, Dox and UCNP@P -Dox / siRNA nanoprobes in 3T3 and MCF-7 cells is shown using fluorescence microscopy (Figures 45 and 47). The fluorescence microscopy results for Dox uptake show that UCNP@P -Dox / siRNA in 3T3 cells provides approximately 1.2-fold higher uptake compared to Dox. In MCF-7 cells, UCNP@P-Dox / siRNA consistently exhibited a 2-fold higher uptake rate compared to Dox after 12 h of incubation, indicating enhanced drug delivery efficiency.

[0266] In 3T3 cells, no significant change in the overall uptake rate was observed (Figure 45). However, after 24 h of incubation, the fluorescence intensity increased relatively less with UCNP@P-Dox / siRNA treatment. In MCF-7 cells, UCNP@P -Dox / siRNA treatment continued to show significantly higher fold-fold ratios compared to Dox (p«0.0001). These results confirm that UCNP@P-Dox / siRNA was taken up more efficiently in MCF-7 cells compared to 3T3 cells and exhibited a superior uptake profile at both time points.

[0267] Internalization into the cytoplasm suggests an uptake profile specific to MCF-7 cells and maintains this stability over time despite low Dox concentration. Viability and uptake studies showed a negative correlation in 3T3 cells (except for the Dox-only treatment group), with limited or no uptake but increased viability over time. In contrast, MCF-7 cells exhibited an increased uptake response and showed greater loss of viability compared to 3T3 cells. A possible explanation for this is that overexpression of GLUT5 in breast cancer cells enhances cellular uptake and toxicity by enabling more effective interaction with the fructose groups on the surface of the nanoprobes.

[0268] Apoptosis studies of bionanoprobes

[0269] In order to understand the apoptotic effects of nanoprobes on MCF-7 and 3T3 cells, cells were treated with UCNP@P-Dox and UCNP@P-Dox / siRNA and compared with untreated groups (Figure 48A). After 24 h of treatment, a significant increase in the percentage of total apoptotic cells was observed at a concentration of 15.62 pg / mL for 3T3 cells in both nanoprobe groups (p<0.0001 for UCNP@P-Dox, p=0.0045 for UCNP@P-Dox / siRNA). In addition, this concentration was determined as the critical threshold point at which the viability rate started to decrease in 3T3 cells. For the same concentration, the presence or absence of siRNA did not create a significant difference between the nanoprobe groups.

[0270] Remarkably, MCF-7 cells treated with both nanoprobes at concentrations of 7.80 pg / mL and 1.90 pg / mL show higher percentage of apoptotic cells compared with the untreated group and3T3 cells with lower concentrations. Especially, the concentration of 7.80 pg / mL showed significant change with the addition of siRNA to UCNP@P-Dox nanoprobe (p<0.0001) (Figure 48A).

[0271] After determining the concentrations suitable for apoptotic behavior for 3T3 and MCF-7 cells, the relative mRNA expression changes for Bcl-2 were evaluated. The initial concentrations were determined as 15.62 pg / mL for 3T3 cells and 7.80 pg / mL for MCF-7 cells. In addition, two consecutive dilutions were performed to understand the dose-dependent effects with anti-Bcl-2 siRNA-loaded nanoprobes (UCNP@P-Dox / siRNA). Figure 48B presents the Bcl-2 mRNA expression changes for both cell types. Only Dox treatment was studied with the concentration eluted by UCNP@P-Dox / siRNA.

[0272] As a result, a dose-dependent relationship was observed with Bcl-2 mRNA levels in 3T3 cells, but there was no statistically significant difference between groups and concentrations. On the other hand, a significant decrease was observed between UCNP@P-Dox and UCNP@P-Dox / siRNA nanoprobes in MCF-7 cells, especially at a concentration of 7.80 pg / mL (p=0.0292) (Figure 48B).

[0273] In addition, relative mRNA expression changes of anti-apoptotic and pro-apoptotic genes such as BCL-X, MCL1, BAD, BAX and NOXA were also evaluated for MCF-7 cells. A decrease in BCL-X and MCL1 mRNA expression levels was observed with UCNP@P-Dox / siRNA treatment, but this decrease was not statistically significant (Figure 49). Despite the accumulation of Bcl-2-mediated apoptosis in MCF-7 cells, no significant change was detected in the expression patterns of pro-apoptotic genes such as BAD, BAX and NOXA (Figure 49).

[0274] Testing of Prepared Theranostic Bionanoprobes with In-Vivo Studies Establishment of the breast tumor model

[0275] Estrofem® (2 mg film-coated tablet, Novo Nordisk), a Danish origin, was used for the 21 -day hormone treatment applied to the animals in the formation of the breast model. During the process, MCF-7 cell injections were injected in physiological serum 4 times in total, twice a week (Figure A). In all animals, cell injections were applied to the fat pad area of the right breast tissue. For histopathological examination, both the right breast tissue (injected with MCF-7 cells) and the left breast tissue were taken together for comparison and possible metastasis evaluation. At the same time, in all groups, 2-4 animals from each group wereimaged with the IVIS Lumina Series III device in order to preserve the sensitivity of nude mice to anesthesia and follow-ups were performed for the treatment groups. Weight was monitored at all stages, and tumor volume was measured and calculated after tumor formation.

[0276] 6-8 week old nude mice were used in model formation. 5 experimental animals were used as positive control to test tumor model formation. Tumor presence and survival parameters were followed in the long term. After 21 days of hormone treatment, tumor induction was successfully established, and observational follow-up of the tumor without treatment was carried out until the 45th day.

[0277] Investigation of the Efficacy of Prepared UCNPs in Anticancer Treatment

[0278] In the therapy part of the study, cancer was treated with a dual drug delivery system and the effectiveness of the system in cancer treatment was examined.

[0279] Group lb; negative control group (group where no application was made and no tumor was created). There are 5 animals in this group and no effect is observed in the breast tissue in the following saline injections. The weight change of the animals during the follow-up period is presented in Figure 51 A.

[0280] Group 2b; positive control group (tumor-inducing, untreated group). There are 5 animals in this group. The weight change graph recorded during the follow-up period is shown in Figure 51 A, and the graph showing the average tumor volume change is shown in Figure 5 IB.

[0281] Group 3b; the group that received only Dox as a treatment after tumor model formation. According to the two-way ANOVA analysis; weight change significantly increases with time (p<0.0001), but no statistical significance was found in the change of tumor volume between animals and with time (p=0.6643 among animals and p=0.2583 for each animal with time). The weight change of the animals during the follow-up period is presented in Figure 51 A, and the tumor volume change is presented in Figure 5 IB.

[0282] Group 4b; The group that received only Bcl-2 siRNA as a treatment after tumor model formation. This group was studied with 5 animals. According to the two-way ANOVA analysis; weight change increases significantly with time (p<0.0001). The weight of the animals during follow-up and tumor volume change during the treatment process are presented in Figure 51 A and Figure 5 IB, respectively.Group 5b; the group that received only Bcl-2 siRNA-Dox as a treatment after tumor model formation. This group was completed with 5 animals. According to the two-way ANOVA analysis; weight change (Figure 51 A) did not show a significant change overtime (p=0.1316). Tumor volume change is presented in Figure 5 IB.

[0283] Group 6b; The group that underwent treatment with UCNP@P-Dox after tumor model formation. This group was performed with 10 animals. The weight change obtained from tumor model formation to the end of the treatment follow-up period was found to be statistically significant both in time and between animals (p<0.0001). Figure 51 A presents weight, and Figure 5 IB presents tumor volume changes.

[0284] Group 7b; the group that underwent treatment with UCNP@P / siRNA after tumor model formation. This group was completed with 5 animals. After tumor model formation, the treatment process was initiated with the Dox-free nanoparticle group UCNP@P / siRNA. The weight change from tumor model formation to the end of the treatment follow-up period is shown in Figure 4.85A, and the tumor volume change is shown in Figure 5 IB.

[0285] Group 8b; The group that received UCNP@P -Dox / siRNA as the treatment group after tumor formation was completed with 10 animals. In terms of weight follow-up, the weight of each animal increased overtime until hormone application (p<0.0001) and decreased approximately 10 days after the start of tumor treatment. This decrease is positively correlated with the decrease in tumor volume due to the effectiveness of the treatment. Weight change graphs are shown in Figure 51 A and tumor volume change graphs are shown in Figure 5 IB.

[0286] No significant change in body weight was observed for all treatment groups. During treatment, tumor sizes increased in the control group and tumor growth was slightly suppressed in the free Dox group. Comparably, UCNP@P-Dox had increased tumor suppression. No tumor volume reduction was observed in the siRNA and Dox-siRNA groups compared to free Dox. The tumor volume reduction for the final group, UCNP@P-Dox / siRNA, was found to be considerably higher compared to the other groups. This result demonstrates the superiority of the prepared UCNP@P-Dox / siRNA bionanoprobes over the treatments applied separately and together with siRNA and Dox-loaded dual therapy. It also demonstrates the potential of the siRNA and Dox-loaded dual drug delivery system in treatment (Figure 51 and Figure 53).In vivo Biodistribution Studies

[0287] In order to demonstrate the targeting capacity of the glycopeptide polymer, images taken using the in vivo imaging system (IVIS) at 0, 3, 6, 9, 12, and 24 hours after injection of Dox and UCNP@P-Dox / siRNA groups in tumor-bearing mice show the in vivo biodistribution performance (Figure 50A). When the Dox group was compared with the UCNP@P -Dox / siRNA group, it was determined that UCNP@P-Dox / siRNA accumulated more in the tumor region (Figure 50). This is attributed to passive targeted transport due to the enhanced permeability and retention (EPR) effect as well as active targeting by the GLUT5 targeting units (fructose monomer) present in the polymer structure. In addition, it was observed that UCNP@P-Dox / siRNA not only reached the tumor tissue more effectively, but also was retained in the tumor tissue for a longer time (Figure 50A and B).

[0288] The main organs and tumor tissues from mice sacrificed 24 hours after the injection of Dox and UCNP@P-Dox / siRNA groups were collected and their fluorescence images were examined (Figure 52). Consistent with previous findings, a low level of accumulation was observed in the tumor tissue of the Dox group, while a significant accumulation was detected in the UCNP@P-Dox / siRNA group (p<0.05). In addition, the accumulation of UCNP@P-Dox / siRNA in the heart, liver and kidneys was found to be significantly lower than the others (p<0.05). However, although the accumulation of UCNP@P-Dox / siRNA in the spleen and lung was lower than Dox, the decrease in fluorescence ratio was not statistically significant.

[0289] As a result, it has been shown that the glycopeptide polymer in the structure of the nanoprobe increases the specificity of targeting tumor tissue, reduces accumulation in major organs, and has the potential for bidirectional gene / chemotherapy effects.

[0290] Histopathological Studies

[0291] To more accurately evaluate the antitumor activity of UCNP@P -Dox / siRNA in bidirectional gene and comedotherapy treatments, immunohistochemical analyses including hematoxylin and eosin (H& E) staining and immunostaining for proliferating cell nuclear antigen (PCNA), Bcl-2, and Bax were performed (Figures 54 and 55). In histopathological analysis, no abnormalities were detected in the left breast lobe (control), while nodular or infiltrative tumor foci were detected in the right breast lobe (tumor group). Tumor cells showed pleomorphism, anisocytosis, anisonucleosis, increased nuclei, mitotic figures, and lymphocyte infiltration.In the Dox group, the findings were similar to the tumor group, but a decrease in the number of mitotic figures and some areas of fibrosis were observed. In the UCNP@P-Dox group, more pronounced fibrosis formation and a greater decrease in mitotic figures were observed compared to the Dox group. In the UCNP@P-Dox / siRNA group, fibrosis formation and the presence of necrotic tumor cells became more pronounced compared to the tumor group (Figure 54).

[0292] Mitotic index was found to be significantly higher in the tumor group compared to the treatment groups (p<0.05). However, although there were numerical differences between the treatment groups, no statistically significant difference was observed (p>0.05) (Table 10). PCNA immunostaining showed intense nuclear staining in all groups and this staining was especially observed more prominently in the tumor group (Figure 54). Statistical analysis on PCNA index revealed a significant difference between the tumor group and the other three groups (p<0.05) (Table 10).

[0293] Table 10. Statistical analysis of mitotic index data.

[0294] Group PCNA Mitotic index Tedavi olmami§ 401.75±12.49a2.55±0.55aDox 285±4.95b0.80±0.09bUCNP@P-Dox 218±12.17c0.54±0.12bUCNP@P -Dox / siRNA 192.77±8.13c0.33±0.06b

[0295] P value <0.001 <0.002ab cshow the statistical difference between the groups (p <0.05).

[0296] Double immunofluorescence staining using Bcl-2 and Bax primary antibodies revealed intracytoplasmic immunopositive reactions in tumor cells. Immunofluorescence staining suggests a correlation between Bcl-2 silencing and anti-apoptotic activity. The data presented in Table 11 reveal a negative correlation between Bax and Bcl-2. Compared to the Dox group, the UCNP@P-Dox group decreased the anti-apoptotic Bcl-2 protein levels while increasing the pro-apoptotic Bax protein levels (Fig. 56). This correlation becomes more pronounced in the UCNP@P-Dox / siRNA treated tumor groups.Table 11. Statistical correlation of Bax-Bcl-2 stainings.

[0297] Bcl-2 Bax

[0298] Bcl-2 Pearson Correlation 1 -.922**

[0299] Sig. (2 -tailed).000

[0300] N 24 24

[0301] Bax Pearson Correlation -.922** 1

[0302] Sig. (2 -tailed).000

[0303] N 24 24

[0304]

[0305] The correlation is significant at the 0.01 level (2-tailed).

[0306] In the experimental study, it was determined by immunohistochemical and immunofluorescent methods that Bcl-2 immunohistochemical score was high in the tumor control group and BAX expression was high in the treatment groups. Based on the results of histopathological, immunohistochemical and immunofluorescent staining methods and statistical data, this study clearly shows that this treatment method is more effective than other tested treatment methods in mice treated with UCNP@P-Dox / siRNA treatment protocol. All these findings show that the developed UCNP@P-Dox / siRNA nanoprobe provides effective delivery of both Dox and siRNA to the target tumor region and creates strong tumor inhibitory effects.

[0307] Double immunofluorescence staining results

[0308] In the experimental study, it was determined by immunohistochemical and immunofluorescence methods that BCL-2 immunohistochemical score was high in the tumor control group and BAX expression was high in the treatment groups.

[0309] Based on the results of histopathological, immunohistochemical immunofluorescence staining methods and statistical data, this study clearly showed that the UCNP@P -Dox / siRNA treatment protocol was more effective than other tested treatment methods in subjects.

[0310] NaYF4: Yb / Er, which forms the inner core of the multifunctional theranostic nanoparticles developed within the scope of the project, has been successfully synthesized.

[0311] Multifunctional, endosome-escapeable, high drug / gene carrying capacity, targeted, biocompatible polymer layer P(MiMA-Z>-DopMA-Z>-z / ?rFrMA)-Z>-P(Lys)) / Dox) tetrablockpolymer loaded with anticancer drug Dox was successfully synthesized using controlled polymerization techniques RAFT, ring-opening (ROP) polymerization and click chemistry. For the preparation of theranostic nanoparticles, UCNPs were coated with multifunctional magnetic polymer and characterized.

[0312] The coating of polymer onto UCNPs was characterized by TEM analysis and the polymer coated around the UCNPs was clearly seen.

[0313] The amount of Dox conjugated to the multifunctional polymer coated on nanoparticles was found to be approximately 50%.

[0314] antiBCL2 siRNA to prevent apoptotic resistance of cells. Complex formation was studied using agarose gel running method. Accordingly, the best complexation was found to be (N / P) / siRNA ratio of 5 / 1.

[0315] The pH-controlled release profiles of the multifunctional theranostic system were investigated and found to exhibit pH-controlled Dox release in a targeted manner.

[0316] When theranostic nanoparticles were evaluated in terms of their intracellular uptake potential, it was shown that sugar molecules entered the cell at over 80% as targeted with significant contributions to the system in less than 12 hours, independent of dose titration. Intracellular uptake was successfully achieved at 6 hours in MCF-7 cells.

[0317] The designed bionanoprobes have been targeted especially for apoptosis with a mitochondrial start from cell death pathways. As it is known, one of the two main death pathways of the cell occurs in this organelle. The main role here belongs to the BCL-2 family proteins that are responsible for controlling apoptosis. The specific antiBCL2 siRNA molecule added to the design is the target mitochondria, especially on an organelle basis. The BCL-2 family includes pro-apoptotic and anti-apoptotic genes and proteins that are their products in controlling apoptosis. The cytoplasmic forms of these proteins are normally inactive. Pro-apoptotic signals direct these proteins to the mitochondria. Pro-apoptotic and anti-apoptotic molecules meet on the surface of the mitochondria and if the pro-apoptotic mechanism prevails over the anti-apoptotic mechanism, the apoptosis process begins. Conjugation of nanoparticles with antiBCL2 siRNA targeted mitochondria within the cell, thereby directing pro-apoptotic signals towards apoptosis.

[0318] Silencing of BCL2 via siRNA was proven by the decrease in the expression of both anti-apoptotic MCL1 and BCL-xL. In other words, it was determined by the changes at the gene andprotein level that the pro-apoptotic pathway suppressed the anti-apoptotic pathway. Another evidence of this was the increased expression of pro-apoptotic BAX and BAD.

[0319] The MCL1 molecule is a priority molecule targeted in cancer models resistant to anti-cancer drugs (Wang et al., 2021). Apart from the apoptotic effects we observed, our findings suggest that these designed theranostic nanoparticles are promising for drug-resistant cancer models in terms of their effects on the MCL1 molecule.

[0320] It has also been shown by flow cytometry that changes in apoptotic cell profiles of breast cancer cell lines increase in a specific dose-dependent manner.

[0321] The cell line that most effectively / rapidly directs cancer to apoptosis was planned to be selected as a candidate cancer model in the in vivo model and it was determined that the multifunctional theranostic system successfully provided apoptosis in both breast cancer cell types.

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Claims

CLAIMS1. Nanoprobe production method comprising steps of;a. Synthesizing NaYF^ Yb3+ / Er3+nanocrystals (UCNP) suitable for emission when excited with NIR laser, which forms the core of the bionanoprobe,b. Obtaining citrate-coated NaYF4: Yb3+ / Er3+nanocrystals by keeping them in citrate buffer solution to perform ligand exchange,c. Synthesizing P(MiMA-Z>-DopMA-Z>-FrMA)-Z>-P(Lys) tetrablock copolymer as glycopeptide,d. Loading Doxorubicin onto P(MiMA-Z>-DopMA-Z>-FrMA)-Z>-P(Lys) tetrablock copolymer,e. Preparing nanoprobes by reacting citrate-coated NaYF4: Yb3+ / Er3+nanocrystals with Doxorubicin-loaded P(MiMA-Z>-DopMA-Z>-FrMA)-Z>-P(Lyz) tetrablock copolymer.

2. The method according to claim 1, characterized in that the obtained nanoprobe is given magnetic properties by coating FesCU Nanoparticles.

3. The method according to claim 1, characterized in loading siRNA i into the obtained nanoprobe.

4. The method according to claim 2, characterized in loading siRNA into the magnetic nanoprobe obtained.

5. A nanoprobe prepared according to one of the preceeding claims.

Images