WO2023178878A1 - 一种增强实体瘤内原卟啉ix蓄积的微针贴片及其制备方法 - Google Patents
一种增强实体瘤内原卟啉ix蓄积的微针贴片及其制备方法 Download PDFInfo
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- WO2023178878A1 WO2023178878A1 PCT/CN2022/103330 CN2022103330W WO2023178878A1 WO 2023178878 A1 WO2023178878 A1 WO 2023178878A1 CN 2022103330 W CN2022103330 W CN 2022103330W WO 2023178878 A1 WO2023178878 A1 WO 2023178878A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
- A61M2037/0046—Solid microneedles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
- A61M2037/0053—Methods for producing microneedles
Definitions
- the present invention relates to the field of microneedle patch preparation and biomedicine, and in particular to a microneedle patch that enhances PpIX accumulation in solid tumors and a preparation method thereof.
- 5-Aminolevulinic acid is selectively taken up by tumor cells and rapidly biosynthesized into protoporphyrin IX (PpIX) with fluorescent properties, which can be used for fluorescence imaging-mediated tumor diagnosis. Therefore, the photochemical reaction of 5-ALA-based photodynamic therapy (5-ALA-PDT) mainly occurs in cancer cells, avoiding off-target toxic side effects.
- 5-ALA-PDT Although topical 5-ALA-PDT has become the main treatment for patients with actinic keratosis or basal cell carcinoma, it suffers from the limited intratumoral biosynthesis concentration of PpIX and short tumor residence time, as well as tumor heterogeneity, tumor hypoxic microorganisms and Characteristics such as environment and strong antioxidant system limit the clinical application of 5-ALA-PDT in the treatment of solid malignant tumors.
- the treatment plan of 5-ALA-PDT currently lacks non-invasive molecular imaging guidance, such as: the time point of the maximum PpIX concentration in the tumor, the oxygen concentration in the tumor, the number of laser irradiations and other parameters. Therefore, there is an urgent need to develop more effective precision diagnosis and treatment solutions to solve the clinical bottleneck of 5-ALA-PDT, improve its biological safety, and promote its further clinical application.
- Efficient intratumoral production of PpIX is an important condition for 5-ALA-PDT, and the high polarity and hydrophilicity of 5-ALA limits its penetration through the stratum corneum and cell membranes through passive diffusion, resulting in poor performance in oral and transdermal drug delivery. Extremely low bioavailability of 5-ALA.
- the efficiency of converting 5-ALA taken up by tumor cells into PpIX is crucial because it determines the maximum concentration of PpIX produced within tumors and the time of tumor retention. Due to the Warburg effect of tumor cell respiration, respiratory dysfunction caused by tumor cell glycolysis can lead to an imbalance in the supply of PpIX and Fe 2+ .
- the inhibition of HIF-1 ⁇ expression takes a long time, and PpIX is metabolized quickly in the body (reaching the highest peak of synthesis in about 4 hours and being rapidly cleared within 12 hours), so effective strategies are needed to prolong tumor growth.
- Residence time of PpIX The microneedle delivery system can directly penetrate the stratum corneum for transdermal drug delivery. Nanomedicine delivery systems can achieve sustained release of drugs. Therefore, the present invention designs and synthesizes a microneedle patch, which simultaneously achieves intracellular delivery of 5-ALA and catalase (CAT) by integrating nano-delivery drugs and transdermal drug delivery, thereby upregulating the physiological synthesis of PpIX. concentration and prolong the actual effect of its residence time in the tumor, thereby improving the efficacy of 5-ALA-PDT.
- CAT catalase
- the purpose of the present invention is to provide a microneedle patch that enhances PpIX accumulation in solid tumors and a preparation method thereof, aiming to solve the problems caused by the lack of specific delivery strategy in the existing 5-ALA-PDT.
- the resulting problems include tumor hypoxia, low PpIX synthesis concentration, and low efficiency of solid tumor treatment.
- a method for preparing a microneedle patch that enhances PpIX accumulation in solid tumors including the following steps:
- CCPCA nanoparticles and microneedle matrix solution were loaded into the microneedle mold, and after vacuum drying and demoulding, a microneedle patch that enhanced PpIX accumulation in solid tumors was obtained.
- the step of dissolving albumin into Dulbecco's medium, adding CuCl 2 and CaCl 2 for in-situ mineralization to obtain CCP nanoparticles specifically includes:
- Dissolve albumin in Dulbecco's medium then add CuCl 2 solution, mineralize at 37°C for 24 hours, then add CaCl 2 solution, and continue mineralization at 37°C for 24 hours to obtain CCP nanoparticles.
- the albumin may be human serum albumin (HSA) or bovine serum albumin (BSA).
- HSA human serum albumin
- BSA bovine serum albumin
- the concentration of the CuCl 2 solution is 0.1M
- the concentration of the CaCl 2 solution is 1M
- the added volumes of the CuCl 2 solution and the CaCl 2 solution are the same.
- the step of loading CAT and 5-ALA onto the surface of the CCP nanoparticles through one-step carbodiimide coupling method and electrostatic adsorption method to obtain CCPCA nanoparticles specifically includes:
- the CCPC nanoparticles are dispersed in the 5-ALA solution, and after stirring for a third predetermined time at room temperature and in the dark, the CCPCA nanoparticles are obtained.
- the mass ratio of CAT to CCP nanoparticles is 0 to 0.1
- the mass ratio of 5-ALA to CCP nanoparticles is 0 to 0.1
- the drug loading amount of CAT is 0-7.9%, and the drug loading amount of 5-ALA is 0-6.8%.
- the step of loading CCPCA nanoparticles and microneedle matrix solution into a microneedle mold, and after vacuum drying and demolding, to obtain a microneedle patch that enhances PpIX accumulation in solid tumors specifically includes:
- microneedle molds Provide microneedle molds
- the CCPCA nanoparticle suspension is added to the microneedle mold, vacuum drying is performed, the microneedle matrix solution is injected into the microneedle mold, and after vacuum drying and demoulding, a microneedle that enhances PpIX accumulation in solid tumors is obtained. Needle patch.
- the microneedle matrix solution includes sodium hyaluronate and superactive hyaluronic acid (HA), and the weight ratio of the sodium hyaluronate and superactive hyaluronic acid is 1: (1-5).
- microneedle patch that enhances PpIX accumulation in solid tumors, wherein the microneedle patch includes: a patch and a plurality of microneedles arranged in an array on the patch;
- the patch is loaded with a microneedle matrix
- the microneedles are loaded with CCPCA nanoparticles; wherein the CCPCA nanoparticles include CCP nanoparticles and CAT and 5-ALA loaded on the surface of the CCP nanoparticles;
- the microneedle patch is prepared by the method of the present invention.
- the microneedle matrix includes sodium hyaluronate and superactive hyaluronic acid, and the weight ratio of the sodium hyaluronate and superactive hyaluronic acid is 1:(1-5).
- the present invention achieves the co-delivery capability of efficient and precise transdermal delivery of CAT and 5-ALA through the combination of nano sustained release and microneedle delivery strategies.
- the CCPCA nanoparticles induced PpIX accumulation in A375 melanoma cells (141.4 ⁇ 1.7 ng/10 6 cells).
- the CCPA group (72.4 ⁇ 7.7ng/10 6 cells) and the free 5-ALA group (35.1 ⁇ 3.3ng/10 6 cells) of CAT were 1.9 times and 4.0 times higher.
- the MN-CCPCA microneedle patch based on the nano-therapeutic design of the present invention can significantly increase the accumulation of PpIX in the tumor site, which in subcutaneous U87MG brain glioblastoma
- the maximum fluorescence intensity of internal PpIX was 5.3 times and 2.7 times that of the tail vein injection 5-ALA group and the microneedle delivery 5-ALA group, respectively.
- the maximum fluorescence intensity of PpIX in subcutaneous 4T1 breast cancer cells was 4.3 times and 3.2 times that of the tail vein injection 5-ALA group and the microneedle delivery 5-ALA group, respectively.
- the strategy of the present invention to increase PpIX accumulation is to increase the synthesis of endogenous and exogenous PpIX and to minimize the efflux of PpIX in cancer cells.
- the MN-CCPCA microneedle patch can reverse tumor hypoxia, regulate PpIX-related synthases, and circumvent the negative reaction regulation process of PpIX.
- the molecular mechanism is that CAT catalyzes endogenous hydrogen peroxide in cells to continuously increase O 2 levels and inhibit The expression of HIF-1 ⁇ , in turn, reduces the expression of FECH, thereby limiting the efflux of PpIX in mitochondria and triggering a negative feedback effect of heme shortage, increasing the expression of ALA synthase (ALAS), making the exogenous 5-ALA delivered by CCPCA nanoparticles Endogenous 5-ALA synthesized by cancer cells can be simultaneously converted into PpIX, which further leads to increased accumulation of PpIX in cells.
- ALAS ALA synthase
- the present invention successfully provides a microneedle patch that enhances the accumulation of PpIX in solid tumors.
- tumor hypoxia By reversing tumor hypoxia, it increases the maximum physiological concentration of PpIX synthesized in the tumor site and also prolongs the time for PpIX to reach the maximum accumulation peak (
- the maximum tumor retention time of PpIX in glioblastoma, melanoma, and breast cancer is extended to 12, 12, and 24 hours respectively, which is significantly higher than the 4 hours of free 5-ALA), which can provide a longer therapeutic window and enable It can be used for multiple repeated PDT under the premise of one administration.
- the present invention can formulate personalized treatment plans for a variety of superficial tumors (breast cancer, brain glioblastoma, melanoma), and can provide real-time PpIX fluorescence imaging and photoacoustic blood oxygenation imaging with the help of fluorescence and photoacoustic instruments. guidance, significantly improving the effectiveness of cancer treatment.
- superficial tumors breast cancer, brain glioblastoma, melanoma
- PpIX fluorescence imaging and photoacoustic blood oxygenation imaging with the help of fluorescence and photoacoustic instruments. guidance, significantly improving the effectiveness of cancer treatment.
- Figure 1 is a schematic diagram of the application of a microneedle patch that enhances PpIX accumulation in solid tumors in Example 1.
- a in Figure 2 is a transmission electron microscope image of the CCPCA NPs obtained in Example 1
- b in Figure 2 is a scanning electron microscope image of the MN-CCPCA microneedle patch obtained in Example 1.
- a in Figure 3 is a high-resolution scanning electron microscope image of the MN-CCCPA microneedle patch before loading CCPCA NPs in Example 1
- b in Figure 3 is a high-resolution scanning electron microscope image of the MN-CCCPA microneedle patch in Example 1 after loading CCPCA NPs. Scanning electron micrograph.
- Figure 4 shows the encapsulation efficiency and drug loading capacity of 5-ALA in CCPCA NPs obtained in Example 1.
- Figure 5 is the thermal weight loss curve of CCPA and CCPCA NPs obtained in Example 1.
- Figure 6 shows the quantitative values of the dissolved oxygen produced by CCPCA NPs at different concentrations (0-160 mg/mL) obtained in Example 1 under the condition of 1mM H 2 O 2 .
- Figure 7 is a comparison chart of the catalytic kinetics of 10mM H 2 O 2 catalyzed by CAT in CCPCA NPs obtained in Example 1 and the same amount of CAT in vitro.
- Figure 9 is a mechanical property test chart of the MN-CCPCA microneedle patch obtained in Example 1.
- Figure 10 is a hematoxylin and eosin staining diagram of the MN-CCPCA microneedle patch obtained in Example 1 penetrating the stratum corneum in mouse epidermis (a) and rat epidermis (b).
- Figure 11 shows the quantitative values of PpIX accumulation in cells by different nanoparticles obtained in the examples.
- Figure 12 shows the semi-quantitative values of PpIX fluorescence produced in A375 tumor cells in different groups under the condition of adding an equal amount of 5-ALA in the Example.
- Figure 13 shows the intracellular expression of different groups of related enzymes that regulate PpIX synthesis in the Examples.
- Figure 14 shows the semi-quantitative fluorescence values of PpIX produced by different microneedle groups in living A375 melanoma obtained in the Example.
- Figure 15 shows the real-time quantified values of blood oxygen saturation in living A375 melanoma obtained by different microneedle groups obtained in the Example.
- Figure 16 shows (a) tumor volume inhibition curves and (b) tumor photos obtained by using PDT in vivo A375 melanoma using different microneedle groups obtained in the Example.
- Figure 17 is the fluorescence semi-quantitative value of PpIX produced in living U87MG brain glioblastoma by different administration method groups obtained in the Example.
- Figure 18 shows (a) tumor volume inhibition curves and (b) tumor photos obtained after using PDT on in vivo U87MG brain glioblastoma in different administration method groups.
- Figure 19 shows the fluorescence semi-quantitative values of PpIX produced in 4T1 triple-negative breast tumors in vivo by different administration method groups obtained in the Example.
- Figure 20 shows (a) tumor volume inhibition curves and (b) tumor photos obtained after using PDT in 4T1 triple-negative breast tumors in vivo using different administration method groups.
- the present invention provides a microneedle patch that enhances the accumulation of PpIX in solid tumors and a preparation method thereof.
- a microneedle patch that enhances the accumulation of PpIX in solid tumors and a preparation method thereof.
- Embodiments of the present invention provide a method for preparing a microneedle patch that enhances PpIX accumulation in solid tumors, which includes the following steps:
- the MN-CCPCA has the ability to co-deliver CAT and 5-ALA through transdermal delivery with high efficiency and precision.
- the CCPCA NPs have good weak acidic pH (6.0-7.4) degradation ability and the ability to catalyze hydrogen peroxide (H 2 O 2 ) in tumors to produce O 2 .
- the MN-CCPCA can regulate PpIX-related synthases and circumvent the negative reaction regulatory process of PpIX by reversing tumor hypoxia.
- the specific molecular mechanism is that CAT continues to increase endogenous O2 levels and inhibits the expression of HIF-1 ⁇ .
- the MN-CCPCA described in this example can increase intra-tumoral PpIX and O2 levels through precise delivery of CAT and 5-ALA, while providing real-time dual-modal PpIX fluorescence imaging and photoacoustic blood oxygenation imaging guidance, thereby optimizing the 5-ALA-based
- the PDT parameters of ALA improve the efficacy of PDT on three different cancers (breast cancer, glioblastoma, melanoma) and reduce side effects.
- the albumin may be a protein such as human serum albumin (HSA) or bovine serum albumin (BSA).
- HSA human serum albumin
- BSA bovine serum albumin
- the steps of dissolving albumin into Dulbecco's medium, adding CuCl 2 and CaCl 2 for in-situ mineralization to obtain CCP nanoparticles specifically include:
- Negatively charged HSA tends to intensively adsorb Ca 2+ and Cu 2+ , causing local supersaturation of ions and providing nucleation sites, while the doping of Cu 2+ is beneficial to stabilizing the nucleation of CCPCA nanoparticles and hindering calcium phosphate It transforms from amorphous state to crystalline state, thereby inducing in-situ mineralization and forming nanometer-sized CCP particles.
- the concentration of the CuCl 2 solution is 0.1M
- the concentration of the CaCl 2 solution is 1M
- the added volumes of the CuCl 2 solution and the CaCl 2 solution are the same.
- step S2 in one embodiment, the step of loading CAT and 5-ALA onto the surface of the CCP nanoparticles through a one-step carbodiimide coupling method and an electrostatic adsorption method to obtain CCPCA nanoparticles, specifically. include:
- CCP nanoparticle solution Disperse the CCP nanoparticles in a buffer (such as PBS buffer, adjust the pH to about 7.4) to obtain a CCP nanoparticle solution;
- a buffer such as PBS buffer, adjust the pH to about 7.4
- the CCPC nanoparticles are dispersed in a 5-ALA solution (5-ALA is dispersed in a buffer, the buffer can be a 0.1mM PBS solution, the pH is adjusted to about 7.0), and stirred at room temperature and in the dark for a third predetermined time After a period of time (the time may be 1-3 hours, such as 2 hours), the CCPCA nanoparticles are obtained.
- the mass ratio of CAT to CCP nanoparticles is 0-0.1
- the mass ratio of 5-ALA to CCP nanoparticles is 0-0.1.
- the drug loading amount of CAT is 0-7.9%, and the drug loading amount of 5-ALA is 0-6.8%.
- step S3 a two-step micromolding process is used to load CCPCA nanoparticles and microneedle matrix solution into the microneedle mold.
- the step of loading CCPCA nanoparticles and microneedle matrix solution into a microneedle mold, and after vacuum drying and demolding, to obtain a microneedle patch that enhances PpIX accumulation in solid tumors specifically includes:
- microneedle mold (the material of the microneedle mold can be PDMS);
- the CCPCA nanoparticle suspension is added to the microneedle mold, vacuum drying is performed, the microneedle matrix solution is injected into the microneedle mold, and after vacuum drying and demoulding, a microneedle matrix solution that enhances PpIX accumulation in solid tumors is obtained. Needle patch.
- the microneedle matrix solution includes sodium hyaluronate (the molecular weight may be but is not limited to 5kDa) and superactive hyaluronic acid (the molecular weight may be but is not limited to 100kDa), and the sodium hyaluronate and The weight ratio of super active hyaluronic acid is 1:(1 ⁇ 5).
- Embodiments of the present invention provide a microneedle patch (MN-CCPCA) that enhances PpIX accumulation in solid tumors, wherein the microneedle patch includes: a patch and a plurality of microneedle arrays located on the patch. Needle;
- the patch is loaded with a microneedle matrix
- the microneedles are loaded with CCPCA nanoparticles; wherein the CCPCA nanoparticles include CCP nanoparticles and CAT and 5-ALA loaded on the surface of the CCP nanoparticles;
- microneedle patch is prepared by the method described in the embodiment of the present invention.
- the microneedle matrix includes sodium hyaluronate and superactive hyaluronic acid, and the weight ratio of the sodium hyaluronate and superactive hyaluronic acid is 1: (1-5).
- MN-CCPCA mainly consists of 5-aminolevulinic acid (5-ALA), catalase (CAT), copper calcium phosphate nanoparticles with tumor acid degradation, superactive hyaluronic acid and hyaluronic acid
- the sodium composition can maximize the enrichment of PpIX in tumor sites by regulating PpIX-related synthases.
- the MN-CCPCA can catalyze endogenous hydrogen peroxide to continuously produce oxygen through CAT, significantly reverse tumor hypoxia, and release false signals of "hemoglobin deficiency" by down-regulating HIF-1 ⁇ and FECH, reducing the rate of heme synthesis by PpIX.
- ALAS is upregulated, promoting the synthesis of endogenous and exogenous 5-ALA, and increasing the accumulation of PpIX in tumor sites.
- the combination of microneedle transdermal delivery and nano-sustained release system strategies can extend the maximum tumor retention time of PpIX in glioblastoma, melanoma, and breast cancer to 12 and 12 days, respectively.
- the fluorescence intensity at the maximum enrichment time point increased by 2.2, 2.7, and 3.2 times respectively.
- This MN-CCPCA can monitor tumor oxygen saturation and PpIX real-time metabolic dynamics in vivo through fluorescence imaging and photoacoustic imaging, while optimizing the treatment parameters of a variety of superficial solid tumors to achieve reproducible, efficient and accurate PDT based on 5-ALA-PDT. , has great clinical translation advantages.
- Nanoparticles with different components and functions used in the following examples were prepared according to the following steps:
- Step 1 Dissolve 100 mg HSA in 10 mL of glucose-free Dulbecco's medium, and then add 100 ⁇ L of CuCl 2 solution (concentration is 0.1M, solvent is deionized water). The system was sealed and mineralized at 37°C for 24 hours. Subsequently, 100 ⁇ L of CaCl2 solution (concentration: 1M, solvent: deionized water) was added to the system, and mineralization was continued at 37 °C for 24 h. Afterwards, CCP NPs were separated and collected by centrifugation (12000 rpm, 10 minutes).
- Step 2 Disperse 2mg CCP NPs in 2mL 0.1mM PBS buffer (pH adjusted to 7.4) to obtain a CCP NPs solution, add 2mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide salt Hydrochloride (EDC ⁇ HCl) was directly dissolved in 100 ⁇ L DMSO and added to the above CCP NPs solution. After stirring and activation for 1 hour at room temperature and in the dark, 2 mg N-hydroxysuccinimide (NHS) was dissolved in 100 ⁇ L DMSO and added to the reaction solution after stirring and activation. Then 0.2 mg/mL CAT was added and stirred for 6 hours. The resulting product was centrifuged at 12000 rpm for 10 minutes to separate and collect CCPC NPs.
- EDC ⁇ HCl 1-ethyl-(3-dimethylaminopropyl)carbodiimide salt Hydrochloride
- NHS N-hydroxysuccinimide
- Step 3 2 mg CCPC NPs were then dispersed in 2 mL of 0.1 mg/mL 5-ALA solution (0.1 mM PBS solution, pH adjusted to 7.0), and then stirred at room temperature and in the dark for 2 hours. Subsequently, CCPCA NPs were separated and collected by centrifugation (12000 rpm, 10 minutes).
- MN-CCP, MN-CCPC, MN-CCPA, and MN-CCPCA used in the following examples are prepared according to the following steps:
- nanoparticles i.e., CCP NPs, CCPC NPs, CCPA NPs, CCPCA NPs
- CCP NPs CCPC NPs
- CCPA NPs CCPA NPs
- CCPCA NPs CCPCA NPs
- Example 1 Preparation of a microneedle patch that enhances PpIX accumulation in solid tumors
- CCPC NPs were dispersed in 2 mL of 0.1 mg/mL 5-ALA solution (0.1 mM PBS solution, pH adjusted to 7.0), and then stirred at room temperature and in the dark for 2 h. Subsequently, CCPCA NPs were separated and collected by centrifugation (12000 rpm, 10 min).
- CCPCA NPs were dispersed in 600 ⁇ L deionized water to obtain CCPCA NPs suspension. Take 100 ⁇ L of CCPCA NPs suspension (10 mg mL -1 ) and deposit it on the surface of each PDMS microneedle mold.
- microneedle matrix solution including sodium hyaluronate and superactive hyaluronic acid, the concentration of superactive hyaluronic acid is 180 mg/mL, the weight ratio of sodium hyaluronate and superactive hyaluronic acid is 1:5) was injected into each PDMS microneedle mold, dried at 37°C for 13 hours, demolded, and finally protected from light at room temperature. Store in desiccator.
- Figure 1 is a schematic diagram of the application of a microneedle patch that enhances PpIX accumulation in solid tumors in Example 1.
- the specific application steps are as follows: press the prepared microneedle patch on the mouse tumor, remove the microneedle patch after 10 minutes of application, and use the IVIS fluorescence imaging spectrum system and Vevo LAZR-X photoacoustic imaging to monitor the tumor site PpIX in real time Fluorescence intensity and blood oxygen saturation (sO 2 ). Repeatable laser irradiation was performed based on the aforementioned two parameters, and tumor volume changes were recorded (recorded every two days).
- CAT significantly reverses tumor hypoxia by catalyzing endogenous hydrogen peroxide to continuously produce oxygen in vivo, and by down-regulating HIF-1 ⁇ and FECH, it releases false signals of "hemoglobin deficiency" and reduces heme synthesis by PpIX. rate, while upregulating ALAS, promoting the synthesis of endogenous and exogenous 5-ALA, and further increasing the accumulation of PpIX in tumor sites.
- This MN-CCPCA can monitor real-time PpIX metabolic dynamics and tumor oxygen saturation in vivo through PpIX fluorescence/photoacoustic imaging, while optimizing treatment parameters for a variety of superficial solid tumors (breast cancer, glioblastoma, melanoma) , to achieve repeatable, efficient and accurate PDT based on 5-ALA-PDT.
- superficial solid tumors breast cancer, glioblastoma, melanoma
- Figure 2 a and b are the transmission electron microscope images of the CCPCA NPs and the scanning electron microscope images of MN-CCPCA respectively. It can be seen from the figure that CCPCA NPs have good dispersion and the microneedle array of MN-CCPCA is evenly arranged. From Figure 3, which compares the high-resolution scanning electron microscopy images of CCPCA NPs before and after loading microneedles, it can be seen that the surface of the microneedles changes from smooth to uneven.
- FIG 4 shows the encapsulation efficiency and drug loading capacity of 5-ALA in CCPCA NPs.
- the mass ratio of 5-ALA to CCP is in the range of 0 to 0.1
- the drug loading capacity of 5-ALA is between 0 and 6.8%.
- the dosage ratio of 5-ALA increases, the drug loading capacity increases accordingly.
- Figure 5 shows the thermal weight loss curves of CCPA and CCPCA NPs, which shows that when the mass ratio of CAT to CCP is in the range of 0 to 0.1, the drug loading capacity of CAT is between 0 and 7.9%. As the CAT dosage ratio increases, its drug loading capacity increases. Increase accordingly.
- Figure 6 shows the change over time of different concentrations of CCPCA NPs catalyzing H 2 O 2 to produce dissolved oxygen under the condition of 1mM H 2 O 2 ;
- Figure 7 This is a kinetic comparison diagram of CAT in CCPCA NPs and the same amount of CAT in vitro catalyzing 10mM H 2 O 2 , proving that the activity of CAT in CCPCA NPs is equivalent to the same amount of free CAT.
- FIG. 9 shows the mechanical property test chart of MN-CCPCA, which shows that it can provide a transdermal penetration capability of 0.39 Newtons/needle.
- Figure 10 shows the hematoxylin and eosin staining images of the MN-CCCPA patch penetrating the stratum corneum in (a) mouse epidermis and (b) rat epidermis, suggesting that MN-CCCPA can directly penetrate the cutin of mice and rats. layer, delivering CAT and 5-ALA transdermally.
- Figure 11 shows the quality comparison data of PpIX produced by A375 tumor cells in different groups. Under the condition of giving the same concentration of 5-ALA (10.8 ⁇ g mL -1 ), the intracellular PpIX content of the CCPCA group (141.4 ⁇ 1.7ng/10 6 cells) were 1.9 times and 4.0 times that of the CCPA without CAT loading (72.4 ⁇ 7.7ng/10 6 cells) and the free 5-ALA group (35.1 ⁇ 3.3ng/10 6 cells) respectively.
- the dynamic fluorescence value of PpIX produced by living cells was monitored in real time through high-content fluorescence microscopy.
- the PpIX concentration of the 5-ALA group and the CCPA group reached the maximum value at 6 and 10 hours after hatching, respectively, and then increased with time. gradually decreases.
- the CCPCA group had the highest PpIX intensity at 24 hours, indicating that the design based on nanocarriers extended the exposure time of 5-ALA in MN-CCPCA, and the retention time of PpIX in the cells also prolonged.
- FIG. 13 shows the protein immunoblot expression of the CCPCA group regulating the expression of PpIX-related synthases.
- MN-CCPCA can regulate PpIX-related synthases by reversing tumor hypoxia, and avoid the negative reaction regulation process of PpIX.
- the molecular mechanism is that CAT catalyzes cell Endogenous hydrogen peroxide continues to increase O2 levels, inhibits the expression of HIF-1 ⁇ , and then reduces the expression of FECH, thereby limiting the efflux of PpIX in the mitochondria and triggering a negative feedback effect of heme shortage, increasing the expression of ALAS, resulting in cell Accumulation of PpIX increases.
- Figure 14 shows a microneedle patch that enhances the accumulation of PpIX in solid tumors in Example 1.
- the IVIS spectral system was used to track the differences in the microneedle designs based on different nanocarriers within the tumor. Quantified fluorescence value of PpIX accumulation at time point.
- the maximum fluorescence intensity of PpIX in the A375 subcutaneous tumor model of the MN-CCPCA was 2.2 times that of the MN-CCPA group without CAT loading, proving that simultaneous delivery of CAT and 5-ALA can significantly extend the synthesis efficiency and tumor retention time of PpIX.
- Figure 15 shows the accumulation of blood oxygen saturation (sO 2 ) at different times of administration using a photoacoustic system to track a microneedle patch that enhances PpIX accumulation in solid tumors in the treatment of mouse A375 subcutaneous tumor model in Example 1. quantified value.
- the MN-CCPCA increased the sO2 level in the tumor by 2.3 times within 24 hours.
- the sO2 level of the MN-CCPCA group at 48 hours was still 1.68 times that of the control group.
- Figure 16 is a comparison photo of the tumor volume inhibition contrast curve and tumor size obtained by using three repeated PDTs in the treatment of the mouse A375 subcutaneous tumor model using a microneedle patch that enhances PpIX accumulation in solid tumors in Example 1.
- the specific characterization method is as follows: 30 A375 model tumor mice with tumors of 70mm3 were evenly divided into 6 experimental groups, namely, blank microneedle group, MN-CCP group without CAT and 5-ALA, and MN with CAT. -CCPC group, MN-CCPCA group loaded with CAT and 5-ALA simultaneously, MN-CCPCA(+) group loaded without CAT and irradiated with laser, MN-CCPCA(+) loaded with CAT and 5-ALA simultaneously and irradiated with laser Group.
- the dosage is 50mg/kg CCPCA NPs, 3.4mg/kg 5-ALA (each microneedle patch).
- Mice in the laser treatment group were irradiated with laser with a wavelength of 635 nm for 10 minutes (200 mW/cm 2 ) at 6, 12, and 24 hours respectively.
- FIG 15 it can be seen from the changes in tumor volume that the tumor volume of the mice in the MN-CCPCA(+) group was significantly reduced, showing good PDT efficacy, proving that simultaneously increasing the levels of PpIX and O2 in the tumor can greatly increase PDT efficiency.
- Figure 17 shows the different drug administration results tracked using the IVIS spectrum system in the treatment of mice with a subcutaneous U87MG brain glioblastoma tumor model using a microneedle patch that enhances PpIX accumulation in solid tumors in Example 1.
- Method Fluorescence quantification of PpIX accumulation at different time points within the tumor.
- the maximum fluorescence intensity of PpIX of the MN-CCPCA in subcutaneous U87MG brain glioblastoma was 5.3 times and 2.7 times that of the tail vein injection 5-ALA group and the microneedle delivery 5-ALA group, respectively.
- Figure 18 shows a microneedle patch that enhances PpIX accumulation in solid tumors in the treatment of mouse subcutaneous U87MG brain glioblastoma model in Example 1, obtained by improving the 5-ALA delivery method and using three repeated PDTs.
- the specific characterization method is as follows: 20 U87MG model tumor mice with tumors of 100mm3 were evenly divided into 4 experimental groups, namely the MN(+) group with blank microneedles and laser irradiation, and the tail vein injection of 5-ALA and laser irradiation.
- 5-ALAi.v(+) group MN-5-ALA(+) group using microneedles to deliver 5-ALA and irradiating laser, and MN using nanoparticles to load transdermal microneedles with 5-ALA and CAT and irradiating laser -CCPCA(+) group.
- the dosage is 50mg/kg CCPCA NPs, 3.4mg/kg 5-ALA.
- Mice in the laser treatment group were irradiated with laser with a wavelength of 635 nm for 10 minutes (200 mW/cm 2 ) at 6, 12, and 24 hours respectively. Use a vernier caliper to measure the volume of mouse tumors in each group on different days (0, 2, 4, 6, 8, 10, 12, 14).
- Figure 19 shows the use of a microneedle patch that enhances PpIX accumulation in solid tumors in the treatment of mouse 4T1 breast cancer subcutaneous tumor model in Example 1.
- the IVIS spectrum system was used to track the different administration methods within the tumor. Fluorescence quantification of PpIX accumulation at time points.
- the maximum fluorescence intensity of PpIX in the subcutaneous 4T1 breast cancer subcutaneous tumors of the MN-CCPCA was 4.3 times and 3.2 times that of the tail vein injection 5-ALA group and the microneedle delivery 5-ALA group, respectively.
- Figure 20 shows the tumor volume inhibition contrast curve and PDT obtained by improving the 5-ALA delivery method and using a microneedle patch that enhances PpIX accumulation in solid tumors in the treatment of mouse 4T1 breast cancer subcutaneous tumor model in Example 1. Comparative photos of tumor size.
- the specific characterization method is as follows: 20 4T1 model tumor mice with tumors of 100mm3 were evenly divided into 4 experimental groups, namely the MN(+) group with blank microneedles and irradiation of laser, and the MN(+) group with tail vein injection of 5-ALA and irradiation with laser.
- 5-ALAi.v(+) group MN-5-ALA(+) group using microneedles to deliver 5-ALA and irradiating laser, and MN using nanoparticles to load transdermal microneedles with 5-ALA and CAT and irradiating laser -CCPCA(+) group.
- Mice in the laser treatment group were irradiated with laser with a wavelength of 635 nm for 10 minutes (200 mW/cm 2 ) at 12, 24, and 36 hours respectively.
- the dosage is 50mg/kg CCPCA NPs, 3.4mg/kg 5-ALA.
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Abstract
一种增强实体瘤内原卟啉IX蓄积的微针贴片及其制备方法。该方法包括步骤:将白蛋白溶解至杜氏培养基中,以白蛋白为模板,加入CuCl 2和CaCl 2进行矿化,得到CCP纳米颗粒;分别通过一步碳二亚胺偶联法和静电吸附法将CAT和5-ALA负载至CCP纳米颗粒表面,得到CCPCA纳米颗粒;将CCPCA纳米颗粒和微针基质溶液装载进微针模具中,经真空干燥脱模,得到增强实体瘤内PpIX蓄积的微针贴片。所述微针贴片通过整合纳米磷酸钙载体和透皮给药方式实现CAT和5-ALA的精准递送,增加肿瘤细胞内PpIX和O 2水平,同时提供实时的双模态PpIX荧光成像和光声血氧成像的指导,提高对不同癌症的光动力学治疗效果。
Description
本发明涉及微针贴片制备及生物医药领域,尤其涉及一种增强实体瘤内PpIX蓄积的微针贴片及其制备方法。
5-氨基酮戊酸(5-ALA)被肿瘤细胞选择性摄取并将其迅速生物合成为具有荧光性能的原卟啉IX(PpIX),可以用于荧光成像介导的肿瘤诊断。因此,基于5-ALA的光动力学治疗(5-ALA-PDT)的光化学反应主要发生在癌细胞中,避免了脱靶的毒副作用。虽然局部5-ALA-PDT已成为光化性角化病或基底细胞癌患者的主要治疗手段,但PpIX瘤内生物合成浓度有限且肿瘤滞留时间短,以及肿瘤的异质性、肿瘤乏氧微环境和强抗氧化系统等特点,限制了5-ALA-PDT在实体恶性肿瘤治疗方面的临床应用。另外,在临床实际应用中,5-ALA-PDT的治疗方案目前缺乏无创的分子影像指导,例如:瘤内最大PpIX的浓度的时间点、瘤内的氧气浓度、激光照射次数等参数。因此,迫切需要开发更有效的精准诊疗方案来解决5-ALA-PDT的临床瓶颈,以提高其生物安全性,并推动其进一步临床应用。
瘤内高效地生成PpIX是5-ALA-PDT的重要条件,而5-ALA的高极性和亲水性限制了其通过被动扩散透过角质层和细胞膜,导致在口服和透皮给药中极低的5-ALA生物利用度。另一方面,肿瘤细胞摄取的5-ALA转化为PpIX的效率至关重要,因为它决定了PpIX瘤内生成的最高浓度以及肿瘤滞留的时间。由于肿瘤细胞呼吸的瓦博格效应,肿瘤细胞糖酵解引起的呼吸功能障碍会导致PpIX和Fe
2+供应失衡,研究表明可以通过供应外源性5-ALA、引入铁螯合剂、降低亚铁螯合酶表达、抑制血红素加氧酶活性等不同策略来上调肿瘤细胞内PpIX的生物合成。然而,胞内PpIX浓度的快速增加,会触发血红素合成反馈调节机制,导致PpIX的排出增强并用于合成血红素,使得细胞内PpIX的累积浓度不足,从而达不到理想的肿瘤治疗效果。研究表明抑制原癌的Ras/MEK信号通路可以通过下调缺氧诱导因子(HIF)-1α(HIF-1α)来降低亚铁螯合酶(FECH)的活性,从而增强细胞内PpIX的累积。因此,给肿瘤细胞持续供氧气(O
2),可以 通过调控Ras/MEK信号通路,通过级联下调HIF-1α和FECH来提高细胞内PpIX的累积浓度,同时克服5-ALA-PDT的肿瘤缺氧问题。然而,HIF-1α表达的抑制需要较长的时间,而PpIX在体内的代谢较快(大约在4小时内达到合成的最高峰,在12小时内迅速清除),因此需要有效的策略来延长肿瘤PpIX的滞留时间。微针递送系统可以直接穿透角质层,进行药物透皮递送。纳米药物递送系统可以实现药物的缓释。因此,本发明设计和合成了一种微针贴片,通过整合纳米递送药物和透皮给药方式,同时实现5-ALA和过氧化氢酶(CAT)的胞内递送,从而上调PpIX生理合成浓度并延长其肿瘤内滞留时间的实际效果,从而提高5-ALA-PDT的疗效。
肿瘤内高浓度单线态氧(
1O
2)的生成是5-ALA-PDT发挥治疗作用的关键。然而,在5-ALA-PDT临床试验报告的不良事件中,诸如治疗部位邻近组织的疼痛、水肿和脱皮等,都与
1O
2在正常组织中的高积累有关。这是由于PDT反应过程中产生的
1O
2会引发氧化损伤以及蛋白质和多核苷酸的二次修饰,从而使膜细胞器更加脆弱并进一步诱导细胞凋亡或坏死。为了增强5-ALA-PDT的抗肿瘤疗效并减轻毒副作用,迫切需要更精准的5-ALA-PDT的诊疗方案,尤其需要实时评估肿瘤组织内的PpIX和O
2浓度,并根据无创的分子影像来制定激光照射参数,优化治疗参数。
因此,现有技术还有待于改进和发展。
发明内容
鉴于上述现有技术的不足,本发明的目的在于提供一种增强实体瘤内PpIX蓄积的微针贴片及其制备方法,旨在解决现有5-ALA-PDT中由于缺乏特异性递送策略而导致的肿瘤乏氧,PpIX合成浓度低,实体肿瘤治疗效率低下等的问题。
本发明的技术方案如下:
一种增强实体瘤内PpIX蓄积的微针贴片的制备方法,其中,包括如下步骤:
将白蛋白溶解至杜氏培养基中,加入CuCl
2和CaCl
2进行原位矿化,得到CCP纳米颗粒;
分别通过一步碳二亚胺偶联法和静电吸附法将CAT和5-ALA负载至所述CCP纳米颗粒表面,得到CCPCA纳米颗粒;
将CCPCA纳米颗粒和微针基质溶液装载进微针模具中,经真空干燥脱模后,得到增强实体瘤内PpIX蓄积的微针贴片。
可选地,所述将白蛋白溶解至杜氏培养基中,加入CuCl
2和CaCl
2进行原位矿化,得到CCP纳米颗粒的步骤,具体包括:
将白蛋白溶解在杜氏培养基中,然后加入CuCl
2溶液,在37℃下矿化24小时,随后加入CaCl
2溶液,在37℃下继续矿化24小时,得到CCP纳米颗粒。
可选地,所述白蛋白可以为人血清白蛋白(HSA)或牛血清白蛋白(BSA)。
可选地,所述CuCl
2溶液浓度为0.1M,所述CaCl
2溶液浓度为1M,所述CuCl
2溶液与所述CaCl
2溶液加入的体积相同。
可选地,所述分别通过一步碳二亚胺偶联法和静电吸附法将CAT和5-ALA负载至所述CCP纳米颗粒表面,得到CCPCA纳米颗粒的步骤,具体包括:
将所述CCP纳米颗粒分散于缓冲液中,得到CCP纳米颗粒溶液;
将1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·HCl)溶液加入至所述CCP纳米颗粒溶液中,在室温和黑暗条件下搅拌第一预定时间后,加入N-羟基丁二酰亚胺溶液,进行反应;
向所述反应后体系中加入CAT,搅拌第二预定时间后,得到CCPC纳米颗粒;
将所述CCPC纳米颗粒分散在5-ALA溶液中,在室温和黑暗条件下搅拌第三预定时间后,得到所述CCPCA纳米颗粒。
可选地,所述CCPCA纳米颗粒中,CAT与CCP纳米颗粒的质量比为0~0.1,5-ALA与CCP纳米颗粒的质量比为0~0.1。
可选地,所述CCPCA纳米颗粒中,CAT的载药量在0~7.9%,5-ALA的载药量在0~6.8%。
可选地,所述将CCPCA纳米颗粒和微针基质溶液装载进微针模具中,经真空干燥脱模后,得到增强实体瘤内PpIX蓄积的微针贴片的步骤,具体包括:
提供微针模具;
将CCPCA纳米颗粒分散在水中,得到CCPCA纳米颗粒悬浮液;
将所述CCPCA纳米颗粒悬浮液加入所述微针模具中,进行真空干燥,将微针基质溶液注入到所述微针模具中,经真空干燥脱模后,得到增强实体瘤内PpIX 蓄积的微针贴片。
可选地,所述微针基质溶液包括透明质酸钠和超活性透明质酸(HA),所述透明质酸钠和超活性透明质酸的重量比为1:(1~5)。
一种增强实体瘤内PpIX蓄积的微针贴片,其中,所述微针贴片包括:贴片和位于所述贴片上的多个阵列设置的微针;
所述贴片内装载有微针基质;
所述微针内装载有CCPCA纳米颗粒;其中,所述CCPCA纳米颗粒包括CCP纳米颗粒和负载于所述CCP纳米颗粒表面的CAT和5-ALA;
所述微针贴片采用本发明所述的方法制备得到。
可选地,所述微针基质包括透明质酸钠和超活性透明质酸,所述透明质酸钠和超活性透明质酸的重量比为1:(1~5)。
本发明具有以下有益效果:
1、本发明通过纳米缓释与微针递送策略的结合,实现了高效精准透皮递送CAT和5-ALA的共递送能力。在给与相同浓度5-ALA(10.8μg mL
-1)的条件下,所述CCPCA纳米颗粒在A375黑色素瘤细胞内诱导PpIX蓄积的含量(141.4±1.7ng/10
6个细胞)分别为不负载CAT的CCPA组(72.4±7.7ng/10
6个细胞)和游离5-ALA组(35.1±3.3ng/10
6个细胞)的1.9倍和4.0倍。相比静脉注射或单纯的微针递送5-ALA的方法,本发明基于纳米诊疗设计的MN-CCPCA微针贴片可显著增加肿瘤部位PpIX的积累,其在皮下U87MG脑胶质母·细胞瘤内PpIX最大荧光强度分别为尾静脉注射5-ALA组和微针递送5-ALA组的5.3倍和2.7倍。其在皮下4T1乳腺癌细胞内PpIX最大荧光强度分别为尾静脉注射5-ALA组和微针递送5-ALA组的4.3倍和3.2倍。
2、本发明上调PpIX蓄积的策略为增加内源性和外源性PpIX合成,并最大地减少PpIX在癌细胞的流出。MN-CCPCA微针贴片可通过逆转肿瘤乏氧,调控PpIX相关的合成酶,规避PpIX的负反应调控过程,其分子机制是CAT催化细胞内源性过氧化氢持续升高O
2水平,抑制HIF-1α的表达,进而降低FECH表达,从而限制PpIX在线粒体的流出并引发血红素短缺的负反馈效应,增加ALA合成酶(ALAS)的表达,使得CCPCA纳米颗粒递送的外源性5-ALA和癌细胞合成的内源性5-ALA能够同时转化成PpIX,从而进一步导致细胞中PpIX的积累增加。
3、本发明成功提供了一种增强实体瘤内PpIX蓄积的微针贴片,通过逆转肿瘤乏氧,增加肿瘤部位PpIX合成的最大生理浓度,同时也延长了PpIX达到的最大累积峰值的时间(PpIX在胶质母细胞瘤、黑色素瘤、乳腺癌中的最大肿瘤滞留时间分别延长至12、12、24小时,显著高于游离5-ALA的4小时),可提供更长的治疗窗口,使其在一次给药前提下,可用于多次重复的PDT。
4、本发明可以针对多种浅表肿瘤(乳腺癌,脑胶质母细胞瘤,黑色素瘤)制定个性化治疗方案,可借助荧光和光声仪器,同时提供实时的PpIX荧光成像和光声血氧成像的指导,显著提高癌症的治疗效果。
图1为实施例1中一种增强实体瘤内PpIX蓄积的微针贴片的应用示意图。
图2中a为实施例1中得到的CCPCA NPs的透射电镜图,图2中b为实施例1中得到的MN-CCPCA微针贴片的扫描电镜图。
图3中a为实施例1中MN-CCPCA微针贴片负载CCPCA NPs前的高分辨扫描电镜图,图3中b为实施例1中MN-CCPCA微针贴片负载CCPCA NPs后的高分辨扫描电镜图。
图4为实施例1中得到的CCPCA NPs中5-ALA的包封率和载药量。
图5为实施例1中得到的CCPA和CCPCA NPs的热失重曲线。
图6为实施例1中得到的不同浓度(0~160mg/mL)的CCPCA NPs在1mM H
2O
2条件下催化产生溶解氧的量化值。
图7为实施例1中得到的CCPCA NPs中CAT与体外等量CAT催化10mM H
2O
2的催化动力学对比图。
图8为实施例1中得到的CCPCA NPs在中性(pH=7.4)和弱酸性(pH=6.0)降解释放Ca
2+(a)和Cu
2+(b)的量化曲线。
图9为实施例1中得到的MN-CCPCA微针贴片的力学性能测试图。
图10为实施例1中得到的MN-CCPCA微针贴片在小鼠表皮(a)和大鼠表皮(b)穿透角质层的苏木精伊红染色图。
图11为实施例中得到的不同纳米颗粒在细胞中蓄积PpIX的量化值。
图12为实施例中添加等量5-ALA条件下,不同组在A375肿瘤细胞产生PpIX荧光半定量值。
图13为实施例中不同组调控PpIX合成的相关酶在细胞内的表达情况。
图14为实施例得到的不同微针组在活体A375黑色素瘤中产生PpIX的荧光半定量值。
图15为实施例得到的不同微针组在活体A375黑色素瘤中血氧饱和度的实时量化值。
图16为实施例得到的不同微针组在活体A375黑色素瘤中,利用PDT后,得到(a)肿瘤体积抑制曲线和(b)肿瘤照片。
图17为实施例得到的不同给药方式组在活体U87MG脑胶质母细胞瘤中产生PpIX的荧光半定量值。
图18为实施例得到的不同给药方式组在活体U87MG脑胶质母细胞瘤,利用PDT后,得到的(a)肿瘤体积抑制曲线和(b)肿瘤照片。
图19为实施例得到的不同给药方式组在活体4T1三阴性乳腺肿瘤中产生PpIX的荧光半定量值。
图20为实施例得到的不同给药方式组在活体4T1三阴性乳腺肿瘤中,利用PDT后,得到的(a)肿瘤体积抑制曲线和(b)肿瘤照片。
本发明提供一种增强实体瘤内PpIX蓄积的微针贴片及其制备方法,为使本发明的目的、技术方案及效果更加清楚、明确,以下对本发明进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
本发明实施例提供一种增强实体瘤内PpIX蓄积的微针贴片的制备方法,其中,包括如下步骤:
S1、将白蛋白(作为模板)溶解至杜氏培养基中,加入CuCl
2和CaCl
2进行原位矿化,得到CCP纳米颗粒(记为CCP NPs);
S2、分别通过一步碳二亚胺偶联法和静电吸附法将CAT和5-ALA负载至所述CCP纳米颗粒表面,得到CCPCA纳米颗粒(记为CCPCA NPs);
S3、将CCPCA纳米颗粒和微针基质溶液装载进微针模具中,经真空干燥脱模后,得到增强实体瘤内PpIX蓄积的微针贴片(记为MN-CCPCA)。
本实施例中,所述MN-CCPCA具有高效精准透皮递送CAT和5-ALA的共 递送能力。所述CCPCA NPs具有良好的弱酸性pH(6.0~7.4)降解能力,催化肿瘤内过氧化氢(H
2O
2)产生O
2的能力。所述MN-CCPCA可通过逆转肿瘤乏氧,调控PpIX相关的合成酶并规避PpIX的负反应调控过程,其具体分子机制是CAT持续升高内源性O
2水平,抑制HIF-1α的表达,进而降低FECH表达,从而限制PpIX在线粒体的流出并引发血红素短缺的负反馈效应,增加ALAS的表达,导致线粒体中PpIX的积累增加。本实施例所述MN-CCPCA可以通过CAT和5-ALA的精准递送增加肿瘤内PpIX和O
2水平,同时提供实时的双模态PpIX荧光成像和光声血氧成像的指导,从而优化基于5-ALA的PDT参数,提高对三种不同(乳腺癌、胶质母细胞瘤、黑色素瘤)癌症的PDT疗效,并降低副作用。
步骤S1中,在一种实施方式中,所述白蛋白可以为人血清白蛋白(HSA)或牛血清白蛋白(BSA)等蛋白。
在一种实施方式中,将白蛋白溶解至杜氏培养基中,加入CuCl
2和CaCl
2进行原位矿化,得到CCP纳米颗粒的步骤,具体包括:
将HSA溶解在无糖的杜氏培养基中,然后加入CuCl
2溶液(溶剂为去离子水),在37℃下矿化24小时,随后加入CaCl
2溶液(溶剂为去离子水),在37℃下继续矿化24小时,得到CCP纳米颗粒。带负电荷的HSA倾向于集中吸附Ca
2+和Cu
2+,造成离子局部过饱和并提供成核位点,而Cu
2+的掺杂入有利于稳定CCPCA纳米颗粒形核,并阻碍磷酸钙由无定型态向晶态转变,从而诱导其原位矿化,形成纳米尺寸的CCP颗粒。
在一种实施方式中,所述CuCl
2溶液浓度为0.1M,所述CaCl
2溶液浓度为1M,所述CuCl
2溶液与所述CaCl
2溶液加入的体积相同。
步骤S2中,在一种实施方式中,所述分别通过一步碳二亚胺偶联法和静电吸附法将CAT和5-ALA负载至所述CCP纳米颗粒表面,得到CCPCA纳米颗粒的步骤,具体包括:
将所述CCP纳米颗粒分散于缓冲液(如PBS缓冲液,pH调至约7.4)中,得到CCP纳米颗粒溶液;
将EDC·HCl溶液(溶剂可以为二甲基亚砜)加入至所述CCP纳米颗粒溶液中,在室温和黑暗条件下搅拌第一预定时间(1~2小时)后,加入N-羟基丁二酰亚胺溶液(溶剂可以为二甲基亚砜)反应(时间可以为5~15分钟,如10分钟), 随后加入CAT溶液(浓度约为0.2mg/mL,溶剂为去离子水),搅拌第二预定时间(时间可以为5~7小时,如6小时)后,得到CCPC纳米颗粒;
将所述CCPC纳米颗粒分散在5-ALA溶液(5-ALA分散于缓冲液中,该缓冲液可以为0.1mM PBS溶液,pH调节至约7.0)中,在室温和黑暗条件下搅拌第三预定时间(时间可以为1-3小时,如2小时)后,得到所述CCPCA纳米颗粒。
在一种实施方式中,所述CCPCA纳米颗粒中,CAT与CCP纳米颗粒的质量比为0~0.1,5-ALA与CCP纳米颗粒的质量比为0~0.1。
在一种实施方式中,所述CCPCA纳米颗粒中,CAT的载药量在0~7.9%,5-ALA的载药量在0~6.8%。
步骤S3中,使用两步微成型工艺将CCPCA纳米颗粒和微针基质溶液装载进微针模具中。在一种实施方式中,所述将CCPCA纳米颗粒和微针基质溶液装载进微针模具中,经真空干燥脱模后,得到增强实体瘤内PpIX蓄积的微针贴片的步骤,具体包括:
提供微针模具(所述微针模具的材质可以为PDMS);
将CCPCA纳米颗粒分散在水中,得到CCPCA纳米颗粒悬浮液;
将所述CCPCA纳米颗粒悬浮液加入所述微针模具中,进行真空干燥,将微针基质溶液注入到所述微针模具中,经真空干燥脱模后,得到增强实体瘤内PpIX蓄积的微针贴片。
在一种实施方式中,所述微针基质溶液包括透明质酸钠(分子量可以为但不限于5kDa)和超活性透明质酸(分子量可以为但不限于100kDa),所述透明质酸钠和超活性透明质酸的重量比为1:(1~5)。
本发明实施例提供一种增强实体瘤内PpIX蓄积的微针贴片(MN-CCPCA),其中,所述微针贴片包括:贴片和位于所述贴片上的多个阵列设置的微针;
所述贴片内装载有微针基质;
所述微针内装载有CCPCA纳米颗粒;其中,所述CCPCA纳米颗粒包括CCP纳米颗粒和负载于所述CCP纳米颗粒表面的CAT和5-ALA;
所述微针贴片采用本发明实施例所述的方法制备得到。
在一种实施方式中,所述微针基质包括透明质酸钠和超活性透明质酸,所述 透明质酸钠和超活性透明质酸的重量比为1:(1~5)。
本实施例中,MN-CCPCA主要由5-氨基酮戊酸(5-ALA),过氧化氢酶(CAT),具有肿瘤酸降解的磷酸铜钙纳米颗粒,超活透明质酸和透明质酸纳构成,可通过调控PpIX相关合成酶,最大限度地在肿瘤部位富集PpIX。该MN-CCPCA可通过CAT催化内源性过氧化氢持续产氧,显著逆转肿瘤缺氧,并通过下调HIF-1α和FECH,释放“血红蛋白缺乏”的假信号,降低PpIX合成血红素的速率,同时上调ALAS,促进内源性和外源性5-ALA的合成,提高PpIX在肿瘤部位的蓄积。相比单纯微针递送5-ALA,通过微针透皮给药和纳米缓释系统的策略结合,分别延长PpIX在胶质母细胞瘤、黑色素瘤、乳腺癌中的最大肿瘤滞留时间至12、12、24小时,最大富集时间点的荧光强度分别提高2.2、2.7、3.2倍。该MN-CCPCA可以通过荧光成像和光声成像监测体内肿瘤氧饱和度和PpIX实时代谢动力学,同时优化多种浅层实体瘤的治疗参数,实现可重复的基于5-ALA-PDT高效精准的PDT,具有极大的临床转化优势。
下面通过具体的实施例对本发明作进一步地说明。
下述实施例所用不同成分和功能的纳米粒子按如下步骤制得:
步骤1:将100mg HSA溶解在10mL不含葡萄糖的杜氏培养基中,然后加入100μL CuCl
2溶液(浓度为0.1M,溶剂为去离子水)。将该系统密封并在37℃下矿化24小时。随后,将100μL CaCl
2溶液(浓度为1M,溶剂为去离子水)添加到系统中,并在37℃下继续矿化24小时。之后,通过用离心(12000rpm,10分钟),分离收集得到CCP NPs。
步骤2:将2mg CCP NPs分散于2mL 0.1mM PBS缓冲液(pH调至7.4)中,得到CCP NPs溶液,将2mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·HCl)直接溶于100μL DMSO中,加入到上述CCP NPs溶液中。在室温和黑暗条件下搅拌激活1小时后,将2mgN-羟基丁二酰亚胺(NHS)溶于100μL DMSO中,加入搅拌激活后的反应液中。然后加入0.2mg/mL CAT搅拌6小时,所得产物经12000rpm离心10分钟,分离收集得到CCPC NPs。
步骤3:随后将2mg CCPC NPs分散在2mL的0.1mg/mL 5-ALA溶液(0.1mM PBS溶液,pH调节至7.0)中,然后在室温和黑暗条件下搅拌2小时。随后通过离心(12000rpm,10分钟),分离和收集得到CCPCA NPs。
同时,在其它相同的条件下,只采取步骤1,3,得到CCPA NPs。
下述实施例所用MN-CCP,MN-CCPC,MN-CCPA,MN-CCPCA按如下步骤制得:
分别将对应的不同纳米颗粒(即CCP NPs、CCPC NPs、CCPA NPs、CCPCA NPs)分散在600μL去离子水中,得到纳米颗粒悬浮液。取100μL纳米颗粒悬浮液(10mg mL
-1)加入到每个PDMS微针模具内,然后置于真空干燥箱中,连续抽真空5次(每次3分钟)后,将800μL的微针基质溶液(包括透明质酸钠和超活性透明质酸,超活性透明质酸浓度为180mg/mL,所述透明质酸钠和超活性透明质酸的重量比为1:5)注入到每个PDMS微针模具内,37℃下干燥13小时脱模,最后在室温下避光储存在干燥器中。
实施例1:一种增强实体瘤内PpIX蓄积的微针贴片的制备
将100mg HSA溶解在10mL不含葡萄糖的杜氏培养基中,然后加入100μL CuCl
2溶液(浓度为0.1M,溶剂为去离子水)。将该系统密封并在37℃下矿化24小时。随后,将100μL CaCl
2溶液(浓度为1M,溶剂为去离子水)添加到系统中,并在37℃下继续矿化24小时。之后,通过离心(12000rpm,10分钟)分离得到CCP NPs。
取2mg CCP NPs分散于2mL 0.1mM磷酸盐缓冲液(PBS,pH调至7.4)中,得到CCP NPs溶液,将2mg EDC·HCl直接溶于100μL DMSO中,加入到上述CCP NPs溶液中。在室温和黑暗条件下搅拌激活1小时后,将2mg N-羟基丁二酰亚胺(NHS)溶于100μL DMSO中后,加入搅拌激活后的反应液中反应10分钟。然后加入CAT溶液(浓度为0.2mg/mL,溶剂为去离子水)搅拌6小时,所得产物经12000rpm离心10分钟,分离收集得到CCPC NPs。
将2mg CCPC NPs分散在2mL的0.1mg/mL 5-ALA溶液(0.1mM PBS溶液,pH调节至7.0)中,然后在室温和黑暗条件下搅拌2小时。随后,通过离心(12000rpm,10分钟)分离和收集得到CCPCA NPs。
将CCPCA NPs分散在600μL去离子水中,得到CCPCA NPs悬浮液。取100μL的CCPCA NPs悬浮液(10mg mL
-1)沉积在每个PDMS微针模具的表面上。将该装置置于真空干燥箱中,连续抽真空5次(每次3分钟)后,将800μL的微针基质溶液(包括透明质酸钠和超活性透明质酸,超活性透明质酸浓度为180mg/mL,所述透明质酸钠和超活性透明质酸的重量比为1:5)注入到每个PDMS微针模具内,37℃下干燥13小时后脱模,最后在室温下避光储存在干燥器中。
图1为本实施例1中一种增强实体瘤内PpIX蓄积的微针贴片的应用示意图。具体应用步骤如下:将制备好的微针贴片压在小鼠肿瘤处,应用10分钟后移除微针贴片,利用IVIS荧光成像光谱系统和Vevo LAZR-X光声成像实时监测肿瘤部位PpIX的荧光强度和血氧饱和度(sO
2)。并根据前述两种参数进行可重复的激光照射,记录肿瘤体积变化(每两天进行一次记录)。
结合图1所示,CAT在体内通过催化内源性过氧化氢持续产氧,显著逆转肿瘤缺氧,并通过下调HIF-1α和FECH,释放“血红蛋白缺乏”的假信号,降低PpIX合成血红素的速率,同时上调ALAS,促进内源性和外源性5-ALA的合成,进一步提高PpIX在肿瘤部位的积累。这种MN-CCPCA可以通过PpIX荧光/光声成像监测体内PpIX实时代谢动力学和肿瘤氧饱和度,同时优化多种浅层实体瘤(乳腺癌、胶质母细胞瘤、黑色素瘤)的治疗参数,实现基于5-ALA-PDT的可重复的高效精准的PDT。
图2中a和b分别为所述CCPCA NPs的透射电镜图和MN-CCPCA的扫描电镜图。从图可以看出CCPCA NPs具有良好的分散性,MN-CCPCA的微针阵列排列均匀。从图3对比CCPCA NPs装载微针前后的高分辨扫描电镜图可以看出,微针表面由光滑变为凹凸不平状。
接着研究了CCPCA NPs的一些基本性质,图4为CCPCA NPs中5-ALA的包封率和载药量。5-ALA与CCP的质量比为0~0.1区间时,5-ALA的载药量在0~6.8%,随着5-ALA投药比的增大,其载药量相应增大。图5为CCPA和CCPCA NPs的热失重曲线,表明CAT与CCP的质量比为0~0.1区间时,CAT的载药量在0~7.9%,随着CAT投药比的增大,其载药量相应增大。
接着对CCPCA NPs中CAT催化H
2O
2产生O
2能力进行验证,图6为在1mM H
2O
2条件下,不同浓度CCPCA NPs催化H
2O
2产生溶解氧随时间的变化情况;图7为CCPCA NPs中CAT与体外等量CAT催化10mM H
2O
2的动力学对比图,证明CCPCA NPs中CAT的活性与等量的游离CAT相当。
接着对CCPCA NPs的酸响应降解能力进行了验证,图8中a、b为得到的CCPCA NPs在中性(pH=7.4)和弱酸性(pH=6.0)降解释放Ca
2+和Cu
2+的量化曲线。将1mL CCPCA NPs溶液(浓度为1mg/mL,溶剂为去离子水)放置于透析袋,并浸入pH值分别为6.0、7.4的9mL磷酸盐缓冲液中,每隔一段时间取1mL溶液用于检测,并同时向该缓冲液中补充1mL对应pH值的磷酸盐缓冲 液。如图8所示,CCPCA NPs在pH 6.0条件下表现出更好的Ca
2+和Cu
2+释放效果,显示出极好的酸响应降解性能。
接着研究了MN-CCPCA的透皮递送能力。图9为MN-CCPCA的力学性能测试图,表明可提供0.39牛顿/针的透皮穿透能力。图10为MN-CCPCA贴片在(a)小鼠表皮和(b)大鼠表皮穿透角质层的苏木精伊红染色图,提示MN-CCPCA可以直接穿透小鼠和大鼠的角质层,透皮递送CAT和5-ALA。
然后研究了MN-CCPCA在细胞和活体肿瘤中蓄积PpIX的生物效应。图11为不同组在A375肿瘤细胞产生PpIX的质量对比数据,在给与相同浓度5-ALA(10.8μg mL
-1)的条件下,所述CCPCA组细胞内PpIX含量(141.4±1.7ng/10
6个细胞)分别为不负载CAT的CCPA(72.4±7.7ng/10
6个细胞)和游离5-ALA组(35.1±3.3ng/10
6个细胞)的1.9倍和4.0倍。
然后通过高内涵荧光显微镜实时监测了活体细胞生成PpIX的动态荧光值,如图12所示,5-ALA组和CCPA组的PpIX浓度分别在孵卵后6和10小时达到最大值,然后随着时间的推移逐渐降低。CCPCA组在24小时PpIX强度最大,表明基于纳米载体的设计使MN-CCPCA中5-ALA的暴露时间延长,PpIX在细胞内滞留时间也随之延长。
图13为CCPCA组调控PpIX相关合成酶表达的蛋白免疫印迹表达情况,MN-CCPCA可通过逆转肿瘤乏氧,调控PpIX相关的合成酶,规避PpIX的负反应调控过程,其分子机制是CAT催化细胞内源性过氧化氢持续升高O
2水平,抑制HIF-1α的表达,进而降低FECH表达,从而限制PpIX在线粒体的流出并引发血红素短缺的负反馈效应,增加ALAS的表达,导致细胞中PpIX的积累增加。
图14为本实施例1一种增强实体瘤内PpIX蓄积的微针贴片在小鼠A375皮下瘤模型治疗中,使用IVIS光谱系统追踪得出的基于不同纳米载体设计的微针在肿瘤内不同时间点PpIX累积的荧光量化值。所述MN-CCPCA在A375皮下瘤模型中的PpIX最大荧光强度为不负载CAT的MN-CCPA组的2.2倍,证明同时递送CAT和5-ALA可显著延长PpIX的合成效率和滞瘤时间。
图15为本实施例1一种增强实体瘤内PpIX蓄积的微针贴片在小鼠A375皮下瘤模型治疗中,使用光声系统追踪的给药不同时间血氧饱和度(sO
2)累积的量化值。所述MN-CCPCA与空白微针对照组相比,在24小时内,MN-CCPCA使肿瘤中sO
2水平提高了2.3倍。即便在24小时进行激光照射消耗氧气后, MN-CCPCA组在48小时sO
2水平仍是对照组的1.68倍,
图16为本实施例1一种增强实体瘤内PpIX蓄积的微针贴片在小鼠A375皮下瘤模型治疗中,利用3次重复的PDT得到的肿瘤体积抑制对比曲线和肿瘤大小的对比照片。其具体表征方式为:将30只肿瘤为70mm
3的A375模型肿瘤小鼠平均分为6个实验组,即空白微针组、不载CAT和5-ALA的MN-CCP组、载CAT的MN-CCPC组、同时载CAT和5-ALA的MN-CCPCA组、未载CAT并照射激光的MN-CCPA(+)组,同时载CAT和5-ALA并照射激光的的MN-CCPCA(+)组。给药剂量为50mg/kg CCPCA NPs,3.4mg/kg 5-ALA(每枚微针贴片)。对激光治疗组的小鼠用波长635nm的激光分别在6,12,24h照射10分钟(200mW/cm
2)。用游标卡尺量取在不同天数(0,2,4,6,8,10,12,14)各组中老鼠肿瘤的体积。如图15所示,从肿瘤体积变化可以看出,MN-CCPCA(+)组小鼠肿瘤体积明显减小,表现出良好的PDT疗效,证明同时提高肿瘤内PpIX和O
2水平可极大地增加PDT效率。
图17为本实施例1一种增强实体瘤内PpIX蓄积的微针贴片在小鼠在小鼠皮下U87MG脑胶质母细胞瘤瘤模型治疗中,使用IVIS光谱系统追踪得出的不同给药方式在肿瘤内不同时间点PpIX累积的荧光量化。所述MN-CCPCA在皮下U87MG脑胶质母细胞瘤内PpIX最大荧光强度分别为尾静脉注射5-ALA组和微针递送5-ALA组的5.3倍和2.7倍。
图18为本实施例1一种增强实体瘤内PpIX蓄积的微针贴片在小鼠皮下U87MG脑胶质母细胞瘤模型治疗中,通过改进5-ALA递送方法,利用3次重复的PDT得到的肿瘤体积抑制对比曲线和肿瘤大小的对比照片。其具体表征方式为:将20只肿瘤为100mm
3的U87MG模型肿瘤小鼠平均分为4个实验组,即空白微针并照射激光的MN(+)组、尾静脉注射5-ALA并照射激光的5-ALAi.v(+)组、微针递送5-ALA并照射激光的MN-5-ALA(+)组、利用纳米颗粒负载5-ALA和CAT的透皮微针并照射激光的MN-CCPCA(+)组。给药剂量为50mg/kg CCPCA NPs,3.4mg/kg 5-ALA。对激光治疗组的小鼠用波长635nm的激光分别在6、12、24小时照射10分钟(200mW/cm
2)。用游标卡尺量取在不同天数(0,2,4,6,8,10,12,14)各组中老鼠肿瘤的体积。如图15所示,从肿瘤体积变化可以计算出,5-ALAi.v(+)组、MN-5-ALA(+)组和MN-CCPCA(+)组的肿瘤生长抑制率分别为19.1%、79.7%和95.2%,MN-CCPCA(+)组 小鼠肿瘤体积明显减小,表现出良好的PDT疗效,表明改进5-ALA递送策略可极大地增加PDT效率。
图19为本实施例1一种增强实体瘤内PpIX蓄积的微针贴片在小鼠4T1乳腺癌皮下肿瘤皮下瘤模型治疗中,使用IVIS光谱系统追踪得出的不同给药方式在肿瘤内不同时间点PpIX累积的荧光量化。所述MN-CCPCA在皮下4T1乳腺癌皮下肿瘤内PpIX最大荧光强度分别为尾静脉注射5-ALA组和微针递送5-ALA组的4.3倍和3.2倍。
图20为本实施例1一种增强实体瘤内PpIX蓄积的微针贴片在小鼠4T1乳腺癌皮下瘤模型治疗中,通过改进5-ALA递送方法,利用PDT的得到肿瘤体积抑制对比曲线和肿瘤大小的对比照片。其具体表征方式为:将20只肿瘤为100mm
3的4T1模型肿瘤小鼠平均分为4个实验组,即空白微针并照射激光的MN(+)组、尾静脉注射5-ALA并照射激光的5-ALAi.v(+)组、微针递送5-ALA并照射激光的MN-5-ALA(+)组、利用纳米颗粒负载5-ALA和CAT的透皮微针并照射激光的MN-CCPCA(+)组。对激光治疗组的小鼠用波长635nm的激光分别在、12、24、36小时照射10分钟(200mW/cm
2)。给药剂量为50mg/kg CCPCA NPs,3.4mg/kg 5-ALA。用游标卡尺量取在不同天数(0,2,4,6,8,10,12,14)各组中老鼠肿瘤的体积。如图20所示,从肿瘤体积变化可以计算出,5-ALA i.v(+)组、MN-5-ALA(+)组和MN-CCPCA(+)组的肿瘤生长抑制率分别为27.5%、51.1%和92.3%,MN-CCPCA(+)组小鼠肿瘤体积明显减小,表现出良好的PDT疗效,表明改进5-ALA递送策略可极大地增加PDT效率。应当理解的是,本发明的应用不限于上述的举例,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (10)
- 一种增强实体瘤内PpIX蓄积的微针贴片的制备方法,其特征在于,包括如下步骤:将白蛋白溶解至杜氏培养基中,加入CuCl 2和CaCl 2进行原位矿化,得到CCP纳米颗粒;分别通过一步碳二亚胺偶联法和静电吸附法将CAT和5-ALA负载至所述CCP纳米颗粒表面,得到CCPCA纳米颗粒;将CCPCA纳米颗粒和微针基质溶液装载进微针模具中,经真空干燥脱模后,得到增强实体瘤内PpIX蓄积的微针贴片。
- 根据权利要求1所述的增强实体瘤内PpIX蓄积的微针贴片的制备方法,其特征在于,所述将白蛋白溶解至杜氏培养基中,加入CuCl 2和CaCl 2进行原位矿化,得到CCP纳米颗粒的步骤,具体包括:将白蛋白溶解在杜氏培养基中,然后加入CuCl 2溶液,在37℃下矿化24小时,随后加入CaCl 2溶液,在37℃下继续矿化24小时,得到CCP纳米颗粒。
- 根据权利要求2所述的增强实体瘤内PpIX蓄积的微针贴片的制备方法,其特征在于,所述白蛋白为人血清白蛋白或牛血清白蛋白;所述CuCl 2溶液浓度为0.1M,所述CaCl 2溶液浓度为1M,所述CuCl 2溶液与所述CaCl 2溶液加入的体积相同。
- 根据权利要求1所述的增强实体瘤内PpIX蓄积的微针贴片的制备方法,其特征在于,所述分别通过一步碳二亚胺偶联法和静电吸附法将CAT和5-ALA负载至所述CCP纳米颗粒表面,得到CCPCA纳米颗粒的步骤,具体包括:将所述CCP纳米颗粒分散于缓冲液中,得到CCP纳米颗粒溶液;将1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐溶液加入至所述CCP纳米颗粒溶液中,在室温和黑暗条件下搅拌第一预定时间后,加入N-羟基丁二酰亚胺溶液,进行反应;向所述反应后体系中加入CAT,搅拌第二预定时间后,得到CCPC纳米颗粒;将所述CCPC纳米颗粒分散在5-ALA溶液中,在室温和黑暗条件下搅拌第三预定时间后,得到所述CCPCA纳米颗粒。
- 根据权利要求1所述的增强实体瘤内PpIX蓄积的微针贴片的制备方法, 其特征在于,所述CCPCA纳米颗粒中,CAT与CCP纳米颗粒的质量比为0~0.1,5-ALA与CCP纳米颗粒的质量比为0~0.1。
- 根据权利要求1所述的增强实体瘤内PpIX蓄积的微针贴片的制备方法,其特征在于,所述CCPCA纳米颗粒中,CAT的载药量在0~7.9%,5-ALA的载药量在0~6.8%。
- 根据权利要求1所述的增强实体瘤内PpIX蓄积的微针贴片的制备方法,其特征在于,所述将CCPCA纳米颗粒和微针基质溶液装载进微针模具中,经真空干燥脱模后,得到增强实体瘤内PpIX蓄积的微针贴片的步骤,具体包括:提供微针模具;将CCPCA纳米颗粒分散在水中,得到CCPCA纳米颗粒悬浮液;将所述CCPCA纳米颗粒悬浮液加入所述微针模具中,进行真空干燥,将微针基质溶液注入到所述微针模具中,经真空干燥脱模后,得到增强实体瘤内PpIX蓄积的微针贴片。
- 根据权利要求1所述的增强实体瘤内PpIX蓄积的微针贴片的制备方法,其特征在于,所述微针基质溶液包括透明质酸钠和超活性透明质酸,所述透明质酸钠和超活性透明质酸的重量比为1:(1-5)。
- 一种增强实体瘤内PpIX蓄积的微针贴片,其特征在于,所述微针贴片包括:贴片和位于所述贴片上的多个阵列设置的微针;所述贴片内装载有微针基质;所述微针内装载有CCPCA纳米颗粒;其中,所述CCPCA纳米颗粒包括CCP纳米颗粒和负载于所述CCP纳米颗粒表面的CAT和5-ALA;所述微针贴片采用权利要求1~8任一项所述的方法制备得到。
- 根据权利要求9所述的增强实体瘤内PpIX蓄积的微针贴片,其特征在于,所述微针基质包括透明质酸钠和超活性透明质酸,所述透明质酸钠和超活性透明质酸的重量比为1:(1-5)。
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