Pharmaceutical composition and application thereof

Abstract

The present application relates to a pharmaceutical composition comprising a hydrophobic substance; a dispersing solvent at a concentration of less than 1 vol%; and a culture medium. Wherein the dispersing solvent is dimethyl sulfoxide at a concentration of 0.1 to 0.9 vol%, and the hydrophobic substance comprises hydrophobic photosensitizers PNA-9, TPE-IQ and CPT. In addition, the present application relates to the use of the above pharmaceutical composition for the preparation of a medicament for photodynamic therapy (PDT). According to the application, in the research on the lipid droplet targeted photosensitizer PNA-9, the low-concentration DMSO (lt, 1% by volume) has obvious influence on the performance of the PDT (photodynamic therapy) of the lipid droplet targeted photosensitizer PNA-9. An in-depth study shows that the low-concentration DMSO has a remarkable regulation effect on the particle size of PNA-9, so that cellular uptake is influenced through gridding protein mediated endocytosis, and the PDT effect is sharply weakened. The invention aims to arouse the attention to the low-concentration DMSO effect and is expected to promote the development of biological materials and the evaluation of the comprehensive performance of the materials.

Description

Technical Field

[0001] This application relates to the field of synthetic pharmaceuticals, and more particularly to a pharmaceutical composition and its use in the preparation of a medicament for photodynamic therapy (PDT). Background Technology

[0002] Organic molecules, as the fundamental building blocks of life and matter, play a crucial role not only in materials science and energy technology but also in modern medicine. Small-molecule drugs derived from organic compounds form the cornerstone of therapeutic systems, with applications spanning multiple fields including cancer, infectious diseases, cardiovascular diseases, and neurological disorders. These drugs have made significant contributions to human health by extending life expectancy, improving quality of life, and advancing precision medicine strategies. Given their profound impact on global health and healthcare systems, the precise evaluation of drug behavior and performance is not only an important topic in basic scientific research but also a responsibility that society must shoulder.

[0003] Currently, commercially available drugs can be broadly categorized into two types based on their origin: natural drugs and synthetic drugs. Many synthetic drugs are strongly hydrophobic and difficult to dissolve directly in aqueous environments; therefore, polar organic solvents such as dimethyl sulfoxide (DMSO) are often used for dissolution in experiments. DMSO has been used as a dispersing solvent in pharmaceutical and life science research for over half a century. Its unique chemical properties enable it to dissolve a wide range of compounds, from small molecule drugs to large polymers, thus finding widespread application in drug preparation, cell experiments, and local drug delivery systems. Almost all research on hydrophobic drugs begins with dissolving the drug in DMSO to prepare a stock solution. Due to its excellent solubility properties, DMSO is known as a "universal solvent." Studies have found that high concentrations of DMSO can enhance cell membrane permeability. For example, research has shown that approximately 10% by volume of DMSO can induce transient aquaporin formation, thereby significantly increasing cell membrane permeability and promoting the transmembrane transport of exogenous molecules. Furthermore, high concentrations of DMSO can induce cytoskeleton remodeling, mitochondrial damage, and apoptosis in various cell lines. In contrast, low concentrations of DMSO (less than 1% by volume) have negligible effects on cell metabolism, membrane stability, and proliferative activity. Therefore, it is generally considered a "safe concentration" and used as a standard solvent reference in drug research and cell experiments. Such low concentrations of DMSO are typically believed to have no effect on drug efficacy, and current research on the effects of low concentrations of DMSO is relatively limited, reflecting a technical bias in existing technology.

[0004] In order to overcome the aforementioned technical bias, the inventors of this application conducted intensive research and unexpectedly discovered that even low concentrations of DMSO, less than 1% by volume, can significantly affect drug efficacy. Summary of the Invention

[0005] The purpose of this application is to overcome the aforementioned technical bias and provide a pharmaceutical composition and its use in the preparation of a medicament for PDT.

[0006] On one hand, this application provides a pharmaceutical composition comprising: Hydrophobic substances; Dispersing solvents with a concentration of less than 1% by volume; Culture medium.

[0007] Preferably, the dispersing solvent is dimethyl sulfoxide.

[0008] Preferably, the dispersing solvent is dimethyl sulfoxide with a concentration of 0.1 to 0.9 vol%.

[0009] Preferably, the dispersing solvent is dimethyl sulfoxide with a concentration of 0.3 to 0.9 vol%, preferably 0.5 to 0.9 vol%.

[0010] Preferably, the hydrophobic substance comprises the hydrophobic photosensitizer PNA-9. TPE-IQ .

[0011] Preferably, the hydrophobic material comprises CPT. .

[0012] Preferably, the culture medium is Gibco's DMEM high-glucose medium, which contains L-glutamate sodium, HEPES, 4.5 g / L glucose, amino acids, vitamins, inorganic salts, phenol red, and does not contain sodium pyruvate.

[0013] On the other hand, this application provides the use of the above-described pharmaceutical composition in the preparation of a medicament for photodynamic therapy (PDT).

[0014] Preferably, the drug is used to treat cancer, infectious diseases, cardiovascular diseases, and nervous system diseases.

[0015] Compared with the prior art, this application overcomes the technical bias that the scientific community generally regards a concentration of less than 1% dimethyl sulfoxide as a "safe concentration", and discovers a pharmaceutical composition containing a concentration of less than 1% dimethyl sulfoxide, which can be used to prepare a drug for photodynamic therapy (PDT) and has a significant impact on the efficacy of PDT. Attached Figure Description

[0016] The present application will now be described in conjunction with the accompanying drawings. In the drawings:

[0017] Figure 1 The chemical structure and photophysical properties of PNA-9 are shown. Among them, Figure 1 a displays PNA-9 (1×10) -5M) UV-Vis absorption spectrum and normalized fluorescence spectrum in PBS (excitation wavelength: 405 nm); Figure 1 b displays PNA-9 (1×10) -5 M) Particle size distribution in a mixed solvent of THF and n-hexane (n-hexane ratio = 90 vol%); Figure 1 c displays PNA-9 (1×10) -5 M) Fluorescence spectra of mixed solvents of THF and n-hexane in different n-hexane ratios; Figure 1 d displays PNA-9 (1×10) -5 M) Maximum emission wavelength in mixed solvents of THF and n-hexane with different n-hexane ratios; Figure 1 e Display illumination (36 mWcm) -2 Under these conditions, PNA-9 (1×10⁻⁶) -5 Whether M) exists or not, DCFH (1×10) -5 The relative fluorescence intensity curve of M at 525 nm; Figure 1 f indicates illumination (36 mW cm) -2 Under these conditions, PNA-9 (1×10⁻⁶) -5 When M) exists or does not exist, HPF (1×10 -5 The relative fluorescence intensity curve of M at 515 nm; Figure 1 g displays illumination (36 mW cm) -2 Under these conditions, PNA-9 (1×10⁻⁶) -5 When M) exists or does not exist, DHR123 (1×10) -5 The relative fluorescence intensity curve of M at 525 nm; Figure 1 h shows the white light irradiation (36 mW cm⁻¹) respectively. -2 Under the condition of ), ABDA (5×10 -5 M) Presence or absence of PNA-9 (1×10⁻⁶) in PBS -5 The relative absorption intensity at M).

[0018] Figure 2 This displays a comparison of the efficacy of PNA-9 photodynamic therapy (PDT). Among them, Figure 2 a shows the cell survival rate of PNA-9 in a dark environment; Figure 2 b shows the cell viability of PNA-9 under light-free conditions (using DMSO); Figure 2 c shows HepG2 cancer cells after PNA-9 (1×10⁻⁹) treatment. -6 After 2 hours of treatment with M), the samples were irradiated with white light (36 mW cm⁻¹) at different DMSO concentrations. -2 The survival status of ); Figure 2d shows HepG2 cancer cells after treatment with PNA-9 (1×10⁻⁶). -6 Two hours after staining with M, the sample was exposed to light (36 mW cm⁻¹). -2 Experimental results of treatment with calcein AM (green, 2×10⁻⁵ minutes): Live cells treated with calcein AM (green, 2×10⁻⁵ minutes) showed the following results: -6 M, 30 minutes) were used to label dead cells, while propidium iodide (PI) (red, 4.5 × 10⁻⁶) was used to label dead cells. -6 M, 5 minutes) staining; Figure 2 e shows the observation of reactive oxygen species (ROS) generation in HepG2 cancer cells using microscopic imaging: cells were first treated with PNA-9 (1×10⁻⁶) at different DMSO volume concentrations. -6 M) was incubated for 2 hours, followed by the addition of DCFH (1×10) -5 M) treatment for 30 minutes, followed by light exposure (36 mW cm) -2 Irradiation (20 minutes).

[0019] Figure 3 This demonstrates the cellular uptake and mechanism of PNA-9. Among them, Figure 3 In cell a, flow cytometry analysis showed that HepG2 cells and PNA-9 (1×10⁻⁶) were correlated. -6 M) Results after incubation for different times; Figure 3 In b, flow cytometry analysis showed that HepG2 cancer cells reacted with PNA-9 (1×10⁻⁶) in culture media containing different concentrations of DMSO. -6 M) Results after 2 hours of incubation; Figure 3 In c, HepG2 cells were exposed to medium containing different concentrations of DMSO for 2 hours to inhibit PNA-9 (1×10⁻⁶ cells / mL). -6 Intake of M); Figure 3 d displays PNA-9 (1×10) -6 M) Particle size distribution when dispersed in 0.1% DMSO in a culture medium; Figure 3 e displays PNA-9 (1×10) -6 M) Particle size distribution when dispersed in 0.9 v / v DMSO in culture medium; Figure 3 f shows the PNA-9 concentration (1×10⁻⁹) at different DMSO concentrations. -6 The average particle size of M); Figure 3 In g, flow cytometry analysis showed that HepG2 cancer cells interacted with multiple endocytosis inhibitors (EIPA: 5×10⁻⁶). -5 M, MβCD: 5×10 -3 After incubating with M for 30 minutes, then with PNA-9 (1×10⁻⁶) -6 M) Results after 1 hour of incubation; Figure 3In h, flow cytometry analysis showed that HepG2 cancer cells, after incubation in ammonium chloride (0.02 M) for 30 minutes, were then incubated with PNA-9 (1×10⁻⁶ mcg). -6 M) Results after 1 hour of incubation; Figure 3 In i, HepG2 cancer cells treated with ammonium chloride (0.02 M) showed improved resistance to PNA-9 (1×10⁻⁶ M) and PNA-9 (1×10⁻⁶ M) levels. -6 Intake of M).

[0020] Figure 4 The results of MTT and DLS experiments on two AIE molecules and three chemotherapeutic drugs are shown. Figure 4 a shows that HepG2 cells were treated with DDTPB (1×10⁻⁶). -5 After treatment with M), the samples were cultured for 1 hour in media containing different concentrations of DMSO, with and without white light irradiation (36 mW cm⁻¹). -2 Cell survival rate (after 30 minutes); Figure 4 b shows the DDTPB (1×10⁻⁶) concentration in culture media containing different concentrations of DMSO. -5 The average particle size of M); Figure 4 c shows HepG2 cancer cells after TPE-IQ (2×10⁻⁶). -6 After treatment with M), the samples were cultured for 1 hour in media containing different concentrations of DMSO, with and without white light irradiation (36 mW cm⁻¹). -2 Cell survival rate (after 30 minutes); Figure 4 d shows the TPE-IQ (2×10) in culture media containing different concentrations of DMSO. -6 The average particle size of M); Figure 4 e shows HepG2 cancer cells after CPT (2.5×10⁻⁶). -6 Cell viability in culture media containing different concentrations of DMSO after M) treatment; Figure 4 f shows the CPT (2.5 × 10⁻⁶) in culture media containing different concentrations of DMSO. -6 The average particle size of M); Figure 4 g showed that HepG2 cancer cells were treated with PTX (1×10⁻⁶). -6 Cell viability in culture media containing different concentrations of DMSO after M) treatment; Figure 4 h shows the levels of PTX (1×10⁻⁶) in culture media containing different concentrations of DMSO. -6 The average particle size of M).

[0021] Figure 5 In Figure 5 a-5h showed PNA-9 at different DMSO concentrations (1×10⁻⁵). -6 M) Flow cytometry analysis of HepG2 cancer cells incubated for 2 hours.

[0022] Figure 6 In Figure 6 a-6c respectively display PNA-9 (1×10^ -6 M) Particle size distribution when dispersed in culture medium in (a) 0.3% DMSO, (b) 0.5% DMSO and (c) 0.7% DMSO.

[0023] Figure 7 In Figure 7 a-7d showed the use of various endocytosis inhibitors (EIPA: 5×10) respectively. -5 M, MβCD: 5 × 10 -3 After incubating HepG2 cancer cells with M for 30 minutes, they were then treated with PNA-9 (1×10⁻⁶). -6 M) Flow cytometry analysis after incubation for 1 hour.

[0024] Figure 8 In Figure 8 a-8c show that HepG2 cancer cells were incubated with NH4Cl (0.02 M) for 30 minutes, and then treated with PNA-9 (1×10⁻⁶ M). -6 M) Flow cytometry analysis after incubation for 1 hour.

[0025] Figure 9 In Figure 9 (a) Display using TPE-IQ (1×10) -6 M) and Figure 9 (b) Display using TPE-IQ (2×10) -6 M) HepG2 cancer cells were treated for 15 minutes at different DMSO concentrations, with or without white light irradiation (36 mW cm⁻¹). -2 Cell survival ability (30 minutes).

[0026] Figure 10 Display using CPT (1×10) -6 The cell survival ability of HepG2 cancer cells treated with M) at different DMSO concentrations.

[0027] Figure 11 In Figure 11 (a) Display using BTZ (5×10) -7 The cell viability of HepG2 cancer cells treated with M) in culture media with different concentrations of DMSO; Figure 11 (b) Display BTZ (5×10) -7 M) Average particle size in media with different concentrations of DMSO. Detailed Implementation

[0028] The advantages and features of this application will be described below with reference to specific implementation schemes, but this application is not limited thereto.

[0029] In the study using the photosensitizer PNA-9 in this application, the inventors discovered that even small changes in DMSO concentration (within the range of <1 vol%) can significantly affect the efficacy of the drug. The structural formula of PNA-9 is shown below:

[0030] Specifically, when the DMSO concentration increased from 0.1% to 0.9% by volume, the cell survival rate of PNA-9-mediated photodynamic therapy (PDT) plummeted from 71% to 21%, demonstrating the significant impact of low-concentration DMSO on the efficacy of PDT. By integrating cell uptake experiments, particle size distribution analysis, and endocytosis pathway studies, the inventors confirmed the crucial role of low-concentration DMSO in regulating drug performance through clathrin-mediated endocytosis. Notably, this effect is not limited to PNA-9 but extends to traditional therapeutic agents such as chemotherapy drugs and other photosensitizers. This discovery challenges the traditional understanding of DMSO as merely a solvent and can provide a new perspective for drug evaluation and clinical application. Photophysical properties of PNA-9 compounds

[0031] The synthesis of the PNA-9 compound was performed according to the method reported in the reference (Zhang, S. et al. Oxygen-independent two-photon photodynamic therapy through novel photoinduced triarylamine-radical cations. Small 21, 2503981 (2025)). The compound exhibits a broad absorption band at 405 nm and an emission peak at 505 nm (see [reference needed]). Figure 1 a) Due to its hydrophobic aromatic ring, PNA-9 is readily soluble in tetrahydrofuran (THF). The solvation ability of this solvent mixture decreases as the proportion of the non-solvent hexane increases. When the hexane component is 90% by volume, PNA-9 forms nanoaggregates with an average diameter of approximately 429.1 nm, a result confirmed by particle size analysis (see [link to relevant documentation]). Figure 1 b). PNA-9 exhibits weak fluorescence in THF solution, but its fluorescence intensity gradually increases with increasing n-hexane concentration (see [link]). Figure 1 c), exhibiting aggregation-induced emission characteristics. Furthermore, PNA-9 also exhibits a solvochromatic effect: when completely dissolved in THF, its maximum emission wavelength is at 515 nm, which undergoes a low-dispersion shift with increasing n-hexane concentration. When the n-hexane component is 90% by volume, the maximum emission wavelength decreases to 474 nm (see c). Figure 1 d).

[0032] To evaluate the photoinduced oxidation ability of PNA-9, this application uses 2',7'-dichlorodihydrofluorescein (DCFH) as an indicator. DCFH itself does not possess fluorescent properties, but it can be oxidized by reactive oxygen species (ROS) to generate the fluorescent product 2',7'-dichlorofluorescein (DCF). In the absence of PNA-9, the fluorescence intensity remained essentially stable under illumination (see [link to PNA-9]). Figure 1 e); while when PNA-9 is exposed to white light (400-800 nm, 36 mW cm⁻¹), ... -2 When ), the fluorescence intensity of DCFH is significantly enhanced (see Figure 1 e). The experimental results clearly confirm that PNA-9 has a significant photo-induced oxidation ability under light conditions.

[0033] To further investigate the source of PNA-9's photoinduced oxidation ability, this application used hydroxyphenylfluorescein (HPF) as an indicator to detect its •OH generation ability. Under the same conditions, the fluorescence intensity of HPF was slightly enhanced in the presence of PNA-9 compared to when HPF was used alone (see [link to relevant documentation]). Figure 1 f). This indicates that PNA-9 has some •OH generation ability, but the activity is weak. Furthermore, dihydrorhodamine 123 (DHR123) and 9,10-anthratridimyl-bis(methylene)dimalonic acid (ABDA) were used to detect •O2. - And the formation of 1O2. The fluorescence intensity of DHR123 in the presence of PNA-9 is almost unchanged compared to when it is used alone (see Figure 1 g). Notably, the absorption intensity of ABDA at 378 nm remained essentially stable after 180 seconds of illumination, independent of the presence or absence of PNA-9 (see g). Figure 1 h). The results confirmed the •O2 of PNA-9. - It has a weak ability to generate 1O2. Therefore, although PNA-9 exhibits a significant photoinduced oxidative stress effect, its activity is almost entirely independent of classical type I and type II reactive oxygen species (ROS), which is consistent with previous findings. Effects of low concentration DMSO on photodynamic therapy

[0034] PNA-9's excellent photo-induced oxidation ability makes it an ideal candidate for photodynamic therapy (PDT). To explore the application potential of PNA-9 in PDT, its cytotoxicity was first evaluated using the methylthiazolyldiphenyltetrazolium (MTT) assay (see [link to study]. Figure 2 a). The results showed that when the PNA-9 concentration reached 1×10⁻⁶, -5 At time M, more than 90% of HepG2 cells survived after 24 hours of incubation, indicating that the dark toxicity of PNA-9 was negligible.

[0035] The low dark toxicity of PNA-9 prompted the inventors to further utilize it as a photosensitizer in photodynamic therapy (PDT). Due to the hydrophobic nature and poor water solubility of PNA-9, the inventors used dimethyl sulfoxide (DMSO) to prepare the stock solution to promote dispersion, and controlled its concentration below 1% by volume in the experiments, which conforms to standard operating procedures in biological experiments. During the experiments, the inventors found that even when other variables remained constant, small differences in DMSO concentration (below 1% by volume) still significantly affected the PDT effect of PNA-9. To explore the underlying mechanism, the inventors investigated the effect of DMSO alone on cell viability. After co-incubating HepG2 cells with 0.9% by volume DMSO for 24 hours, no significant difference in cell viability was observed (see [link to relevant documentation]). Figure 2 (b) This ruled out the possibility that DMSO directly affected cell viability. Subsequently, the inventors investigated the PDT effect of PNA-9 dispersed in DMSO media of different concentrations. To ensure that the PNA-9 concentration in all experimental groups was 1×10⁻⁶, [further details were provided]. -6 M, the inventors prepared concentrations of 1.0 × 10 -3 M, 3.3×10 -4 M, 2×10 -4 M, 1.4×10 -4 M and 1.1×10 -4 The DMSO stock solution of M was used, and a specified volume of the stock solution was dispersed in the culture medium (i.e., Gibco's DMEM high-glucose medium, which contains L-glutamate, HEPES, 4.5 g / L glucose, amino acids, vitamins, inorganic salts, phenol red, and does not contain sodium pyruvate) to achieve a concentration of 1×10⁻⁶. -6 The concentration of PNA-9 in M. HepG2 cells were incubated in these media for 2 hours, then subjected to white light (36 mW cm⁻¹). -2 Irradiation for 5 and 10 minutes. Significant differences in cell viability were observed when PNA was dispersed in culture media containing different concentrations of DMSO (see [link to article]). Figure 2 c). Cell viability decreased sharply with increasing DMSO concentration. After 5 minutes of irradiation, the cell viability of the 0.1% DMSO group was 71.3%, while that of the 0.9% DMSO group dropped to 20.8%, a decrease of 70.8%. When the irradiation time was extended to 10 minutes, the corresponding survival rates further decreased to 34.1% and 16.0%, respectively, corresponding to DMSO concentrations of 0.1% and 0.9%.

[0036] To visually observe the photodynamic therapy (PDT) effects of PNA-9 dispersed in different concentrations of DMSO, the inventors used calcein AM (green fluorescent marker for live cells) and propidium iodide (PI, red fluorescent marker for dead cells) to perform live / dead cell staining experiments. Figure 2 As shown in Figure d, PNA-9 cells treated with different concentrations of DMSO exhibited distinct green and red fluorescent regions. Specifically, in the 0.1% (v / v) DMSO group, most cells showed strong green fluorescence with weak red fluorescence, indicating that most cells remained viable; while in the 0.9% (v / v) DMSO group, cells mainly showed red fluorescence, with only a few cells retaining green fluorescence, suggesting a significant decrease in cell viability. These results are highly consistent with the MTT assay data, confirming that when the PNA-9 concentration and irradiation parameters are constant, slightly increasing the DMSO concentration from 0.1% (v / v) to 0.9% (v / v) can significantly improve the PDT efficacy of PNA-9.

[0037] To further investigate the mechanism by which different concentrations of dimethyl sulfoxide (DMSO) affect the efficacy of photodynamic therapy (PDT), this application uses DCFH as a fluorescent probe to detect the generation of intracellular reactive oxygen species (ROS). In the experiment, HepG2 cancer cells were co-incubated with PNA-9 dispersed in 0.1%, 0.5%, and 0.9% DMSO for 2 hours, respectively, followed by incubation with DCFH for 30 minutes and white light irradiation (36 mW cm⁻¹). −2 (20 minutes) (see below) Figure 2 e). The results showed that the intensity of green fluorescence gradually increased with increasing DMSO concentration. Only weak fluorescence was detected when the DMSO concentration was 0.1% (v / v), indicating a low ROS level at this point; the green fluorescence moderately increased when the concentration increased to 0.5% (v / v), suggesting an increase in ROS generation (see [link to relevant documentation]). Figure 4 The strongest fluorescence signal was observed at a DMSO concentration of 0.9 vol%, indicating that PNA-9 exhibits the highest oxidative activity under these conditions. The combined experimental results confirm that higher volumetric concentrations of DMSO (i.e., 0.5 vol% to 0.9 vol%) effectively promote intracellular ROS production in PNA-9 under light irradiation, thereby significantly enhancing its PDT therapeutic effect. Low-concentration DMSO-dependent cellular uptake and its mechanism

[0038] The above observations are both interesting and crucial, revealing not only the significant impact of low-concentration DMSO on therapeutic efficacy but also explaining the differences in experimental results among different operators. This prompted the inventors to explore the underlying mechanisms. Increased intracellular reactive oxygen species levels at higher DMSO concentrations indicate that more PNA-9 enters the cells. Therefore, the inventors evaluated the internalization kinetics of PNA-9 under different incubation conditions using a cellular uptake assay system. HepG2 cancer cells were co-incubated with PNA-9 dispersed in a medium containing 0.9% DMSO for different times, and the fluorescence intensity of the cells was analyzed by flow cytometry (see [link to study]). Figure 3 a). The results showed that PNA-9 was rapidly taken up by cells in the first 3 hours, reaching a plateau phase after about 4 hours (see [reference]). Figure 4 The inventors further investigated the differences in PNA-9 intracellularization at different DMSO concentrations. Furthermore, HepG2 cells exhibited dose-dependent uptake of PNA-9 at DMSO concentrations ranging from 0.1% to 0.9% (v / v) (see [link to relevant documentation]). Figure 3 b and Figure 5 (a-5h). As the DMSO ratio increased, the relative fluorescence intensity compared to the untreated control group increased by 6.87-fold, 8.88-fold, 8.70-fold, 10.42-fold, and 12.43-fold, respectively (see [reference]). Figure 3 c). Notably, the fluorescence intensity of cells in the medium containing 0.9% DMSO was approximately twice that in the medium containing 0.1% DMSO, indicating that increasing the DMSO concentration significantly enhances the uptake of PNA-9 by cells.

[0039] Since the PNA-9 concentration remained constant across all groups, the differences likely stemmed from their dispersion state. The dispersion state of nanoparticles not only affects their stability but also significantly alters their diameter distribution within biological systems, a key factor determining intracellular uptake efficiency. Previous studies have shown that smaller diameter particles are more readily internalized and tend to accumulate in the cytoplasm. Conversely, poorly aggregated or dispersed particles typically exhibit a larger effective diameter, which reduces cellular uptake efficiency. To delve deeper into the underlying causes of these differences in cellular uptake, the effect of dimethyl sulfoxide (DMSO) on the dispersion state of PNA-9 must be investigated. Notably, as the DMSO proportion increased from 0.1% (v / v) (see [reference missing]), the dispersion state of PNA-9 changed. Figure 3 d) Increase to 0.9% by volume (see Figure 3 e), the average particle size of PNA-9 decreased significantly from 656.8 nm to 260.3 nm, with an overall particle size reduction of approximately 60% (see [reference]). Figure 3 f). The particle size reduction was particularly significant, reaching 49%, when the DMSO content increased from 0.1 vol% to 0.3 vol%. This significant decrease at low DMSO concentrations indicates that the initial increase in DMSO content played a more crucial role in promoting particle deagglomeration and particle size reduction (see f). Figure 6 (a-6c). Experimental results confirmed that low concentrations of DMSO altered the dispersion of PNA-9 and significantly reduced its particle size, indicating that the improved cellular uptake efficiency was mainly attributed to the change in particle size distribution.

[0040] The internalization of nanoparticles primarily relies on endocytosis. This process typically begins with localized invagination or folding of the plasma membrane. Subsequently, the membrane gradually buds inward and forms vesicles, which eventually develop into endocytic vesicles in the cytoplasm. This mechanism not only ensures efficient uptake of extracellular substances but is also the main pathway for nanoparticles to enter cells. In almost all eukaryotic cells, endocytosis can generally be divided into two main categories: phagocytosis and pinocytosis. Phagocytosis usually occurs in specialized phagocytes and is responsible for uptake of larger particles (>500 nm). In contrast, pinocytosis includes macropinocytosis, clathrin-mediated endocytosis (CME), and pit-dependent endocytosis. Among these, CME is widely considered one of the main pathways for nanoparticle uptake, typically involving particles of 60–300 nm.

[0041] To investigate the cellular uptake mechanism of PNA-9, the inventors employed a variety of endocytosis inhibitors. These inhibitors included ammonium chloride (used to induce cytoplasmic acidification and inhibit CME40), methyl-β-cyclodextrin (MβCD) (used to inhibit pit-dependent endocytosis), and ethyl isopropyl amiloride (EIPA) (used to inhibit giant cell formation). HepG2 cells were pretreated with each endocytosis inhibitor for 30 minutes, followed by co-incubation with PNA-9 dispersed in 0.9% (v / v) DMSO medium for 1 hour. After incubation, the fluorescence intensity of the cells was quantitatively analyzed by flow cytometry (see [link to relevant documentation]). Figure 7 a-7d and Figure 8 (a-8c). For example... Figure 3 As shown in g, compared with the positive control, MβCD treatment resulted in almost no change in fluorescence intensity, while EIPA treatment even resulted in a slight increase in fluorescence intensity, indicating that PNA-9 cellular uptake is largely independent of these two endocytic pathways. In contrast, ammonium chloride treatment resulted in a significant decrease in fluorescence intensity compared with the positive control (see g). Figure 3 Quantitative analysis showed that the cell uptake efficiency of the positive control group (PNA-9 only) and the NH4Cl treatment group was 8.16 times and 5.80 times that of the blank group, respectively, equivalent to a reduction of approximately 29% in uptake after CME treatment (see h). Figure 3 i). Overall, these results indicate that PNA-9 is primarily internalized via CME, which is consistent with the particle size distribution of PNA-9.

[0042] The results clearly demonstrate that even a slight increase in DMSO concentration effectively improves the dispersibility of PNA-9 in aqueous solution, thereby reducing its average hydrodynamic size. The reduction in particle size not only contributes to the stable presence of PNA-9 but also promotes its efficient internalization by cells. Therefore, the enhanced cellular uptake of PNA-9 leads to a significant increase in intracellular reactive oxygen species (ROS) generation under irradiation, thereby improving the efficacy of photodynamic therapy (PDT). These results indicate that solvent conditions play a crucial role in regulating the physicochemical properties of nanomedicine carriers, and this regulation ultimately manifests in their biological performance. Widespread effects of low concentration dimethyl sulfoxide

[0043] Based on these observations, the inventors became interested in an important question: whether this low-concentration DMSO effect can be extended to other hydrophobic photosensitizers. To this end, we selected two previously reported photosensitizers, TPE-IQ and DTTPB, for our experiments.

[0044] DTTPB is a membrane-targeting photosensitizer that can specifically target the plasma membrane of different cell types and the lipid bilayer envelope of viruses. To evaluate the effect of dimethyl sulfoxide (DMSO) concentration on the efficacy of DTTPB photodynamic therapy (PDT), the inventors placed HepG2 cells in a solution containing 1×10⁻⁶ DTTPB. -5 M DTTPB medium (i.e., Gibco's DMEM high-glucose medium, which contains L-glutamate, HEPES, 4.5 g / L glucose, amino acids, vitamins, inorganic salts, phenol red, and does not contain sodium pyruvate) was added with different concentrations of DMSO for 1 hour, followed by 30 minutes of white light irradiation (36 mW cm⁻¹). -2 The results showed that different DMSO concentrations in the culture medium had negligible effects on cell viability (see [link]). Figure 4 a). Dynamic light scattering (DLS) analysis of particle size distribution revealed that the particle size of DDTPB was largely unaffected by DMSO concentration (see [link]). Figure 4 (b) This result is consistent with the data from cell experiments. Overall, a slight increase in DMSO concentration (0.2 v / v–1 v / v) had a limited effect on the dispersibility, particle size, and efficacy of DDTPB.

[0045] TPE-IQ is an isoquinoline-based photosensitizer capable of mitochondrial-targeted imaging and photodynamic therapy (PDT). This application investigated the efficacy of PDT in a medium containing different concentrations of dimethyl sulfoxide (DMSO) (i.e., Gibco's DMEM high-glucose medium containing L-glutamate, HEPES, 4.5 g / L glucose, amino acids, vitamins, inorganic salts, phenol red, and excluding sodium pyruvate). HepG2 cells were treated with TPE-IQ (1×10⁻⁶ cells / year). -6After incubation for 15 minutes (M), the cells were exposed to white light for 15 minutes, and cell viability was assessed using the MTT assay. Cell viability decreased slightly when the DMSO concentration increased from 0.1% to 0.9% (see [link to DMSO information]). Figure 9 a). When the TPE-IQ concentration increases to 2×10 -6 At time M, the decrease in cell viability is more significant (see [reference needed]). Figure 9 b). When the incubation time was further extended to 60 minutes, a significant decrease in cell viability was observed. When the DMSO concentration was increased from 0.1 v / v to 0.9 v / v, cell viability decreased from 77.0% to 30.6% (see [link to DMSO documentation]). Figure 4 c). Dynamic light scattering (DLS) analysis showed that the average particle size of TPE-IQ decreased from 782.8 nm to 288.5 nm (see c). Figure 4 d). These results indicate that higher concentrations of DMSO can significantly improve the dispersibility of TPE-IQ, reduce its particle size, and enhance its PDT efficacy.

[0046] The above results indicate that low concentrations of DMSO primarily affect cellular uptake by interfering with the dispersion of hydrophobic substances. This effect is not limited to photosensitizers but also exists in other hydrophobic substances. Therefore, this application selected three hydrophobic drugs—camptothecin (CPT), paclitaxel (PTX), and bortezomib (BTZ)—and investigated the effects of low concentrations of DMSO on them.

[0047] CPT is a quinoline-based cytotoxic alkaloid, initially isolated from *Camptotheca acuminata*, and exhibits significant anticancer activity against various tumors. Its clinical application is primarily limited by its poor physicochemical properties: the lactone ring structure makes CPT highly unstable under physiological conditions, and its poor water solubility further restricts effective delivery and bioavailability. To investigate the effect of low concentrations of DMSO on the antitumor activity of CPT, the inventors treated HepG2 cells with culture media containing different concentrations of DMSO and assessed cell viability using the MTT assay. When the CPT concentration was 2.5 × 10⁻⁶... -6 At time M, as the DMSO concentration increased from 0.1% to 0.9%, cell viability decreased sharply from 44.4% to 19.4% (see [reference needed]). Figure 4 e). In 1×10 -6 A similar trend was observed at M concentrations, with cell viability decreasing from 52.8% to 29.2% (see [link to M concentration]). Figure 10 Dynamic light scattering analysis showed that increasing the DMSO concentration significantly improved the dispersibility of CPT, reducing its particle size from 1264.2 nm at 0.1 vol% DMSO to 749.9 nm at 0.9 vol% DMSO (see [link to relevant documentation]). Figure 4f). Overall, these results indicate that high concentrations of DMSO (0.1 vol% to 0.9 vol%) enhance the solubility and dispersibility of CPT, resulting in smaller particle sizes and improved antitumor activity, thus exhibiting a synergistic effect with low concentrations of DMSO.

[0048] PTX is a clinically significant diterpenoid anticancer drug, widely used in the treatment of various malignant tumors such as ovarian cancer, breast cancer, and lung cancer due to its potent antitumor activity and relatively low toxicity. However, its clinical application is limited by its extremely poor water solubility. PTX has a large hydrophobic diterpenoid skeleton, with a few hydroxyl and amide groups sterically hindered, resulting in poor solubility and low bioavailability. To evaluate the effect of low concentrations of DMSO on PTX activity, HeLa cells were mixed with 1×10⁻⁶ DMSO. -6 M PTX was incubated in medium containing different concentrations of DMSO (i.e., Gibco's DMEM high-glucose medium, which contains L-glutamate, HEPES, 4.5 g / L glucose, amino acids, vitamins, inorganic salts, phenol red, and phenol red, but does not contain sodium pyruvate). MTT assays showed negligible changes in cell viability (see [link to MTT assay]). Figure 4 g). Furthermore, DLS analysis confirmed that the particle size of PTX remained essentially constant across different DMSO concentrations (see [link]). Figure 4 In summary, low concentrations of DMSO (0.1 vol% to 0.9 vol%) had limited effects on the dispersibility, particle size, and antitumor activity of PTX.

[0049] BTZ is a proteasome inhibitor with significant clinical value and has been widely used as a first-line treatment for hematologic malignancies such as multiple myeloma and mantle cell lymphoma. To evaluate the effect of low-concentration DMSO on BTZ activity, HepG2 cells were treated with 5 × 10⁶ BTZ. -7 MBTZ cells were incubated in media containing different concentrations of DMSO (i.e., Gibco's DMEM high-glucose medium, which contains L-glutamate, HEPES, 4.5 g / L glucose, amino acids, vitamins, inorganic salts, phenol red, and excluding sodium pyruvate). Changes in MTT cell viability were negligible (see [link to relevant documentation]). Figure 11 a). Furthermore, DLS analysis confirmed that the particle size of BTZ remained essentially stable at different DMSO concentrations (see [reference]). Figure 11 b). In summary, low concentrations of DMSO (0.1 vol% to 0.9 vol%) had no significant effect on the dispersibility, particle size, and antitumor activity of BTZ.

[0050] The above results not only reveal the effects of low-concentration DMSO on different hydrophobic materials, but also indicate its differential impact on hydrophobic materials. This reminds us that the effects of low-concentration DMSO should be considered when dealing with hydrophobic materials, which can be of great significance for drug development. discuss

[0051] In summary, this application observed a significant effect of low concentrations of DMSO on the efficacy of hydrophobic drugs. Increasing the DMSO concentration from 0.1 vol% to 0.9 vol% resulted in a sharp decrease in cell viability after co-incubation with PNA-9 from 71.3% to 20.8%. A series of studies revealed that subtle changes in DMSO concentration from 0.1 vol% to 0.9 vol% significantly affected the diffusion behavior and particle size of PNA-9 (from 656.8 nm to 260.3 nm), thereby promoting its uptake by cells via the cell membrane efflux (CME) pathway. This enhanced cellular uptake increased reactive oxygen species (ROS) production and improved photodynamic therapy (PDT) efficacy. This low-concentration DMSO effect has been validated in various hydrophobic materials, including photosensitizers and chemotherapeutic drugs. For example, a slight increase in DMSO concentration improved the solubility and dispersibility of CPT and TPE-IQ, accompanied by a decrease in particle size, promoting cellular uptake and enhancing anticancer effects. For other hydrophobic formulations (such as PTX and DDTPB), a slight increase in DMSO concentration had limited impact on solubility, dispersibility, and anticancer efficacy. Overall, these results reveal the differential effect of low-concentration DMSO on hydrophobic drugs, suggesting the need to pay attention to the effects of low-concentration DMSO when processing hydrophobic formulations. These findings can also explain inconsistent results when different operators use the same formulation. Given the widespread use of hydrophobic drugs, this application provides a new variable to consider in the development and application of such drugs. The inventors further anticipate that these findings will attract increasing attention in future research and stimulate further studies on the effects of secondary variables.

Claims

1. A pharmaceutical composition, characterized in that, It includes: Hydrophobic substances; Dispersing solvents with a concentration of less than 1% by volume; Culture medium.

2. The pharmaceutical composition according to claim 1, characterized in that, The dispersing solvent is dimethyl sulfoxide.

3. The pharmaceutical composition according to claim 2, characterized in that, The dispersing solvent is dimethyl sulfoxide at a concentration of 0.1 to 0.9% by volume.

4. The pharmaceutical composition according to claim 3, characterized in that, The dispersing solvent is dimethyl sulfoxide with a concentration of 0.3 to 0.9 vol%, preferably 0.5 to 0.9 vol%.

5. The pharmaceutical composition according to claim 1, characterized in that, The hydrophobic substance contains the hydrophobic photosensitizer PNA-9. TPE-IQ .

6. The pharmaceutical composition according to claim 1, characterized in that, The hydrophobic material contains CPT. .

7. The pharmaceutical composition according to claim 1, characterized in that, The culture medium is Gibco's DMEM high-glucose medium, which contains L-glutamate sodium, HEPES, 4.5 g / L glucose, amino acids, vitamins, inorganic salts, phenol red, and does not contain sodium pyruvate.

8. Use of a pharmaceutical composition according to any one of claims 1-7 in the preparation of a medicament for photodynamic therapy.

9. The use according to claim 8, characterized in that, The drug is used to treat cancer, infectious diseases, cardiovascular diseases, and nervous system diseases.

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