CN107375943B

CN107375943B - Targeted anti-skin squamous cell carcinoma co-loaded super-deformable liposome and preparation method and application thereof

Info

Publication number: CN107375943B
Application number: CN201710647498.4A
Authority: CN
Inventors: 陈中建; 朱全刚; 安多朋; 沈敏; 石磊; 丁佳宁; 孟鸽飞; 武喜营; 喻琴
Original assignee: SHANGHAI DERMATOLOGY HOSPITAL
Current assignee: SHANGHAI DERMATOLOGY HOSPITAL
Priority date: 2017-08-01
Filing date: 2017-08-01
Publication date: 2020-06-23
Anticipated expiration: 2037-08-01
Also published as: CN107375943A

Abstract

The invention relates to the technical field of medicines, in particular to a co-loading super-deformable liposome for targeted anti-skin squamous cell carcinoma, and a preparation method and application thereof.A peptide aptamer rS3-PA mediated photosensitizer ALA and HIF-1 α siRNA combined co-loading super-deformable liposome is firstly constructed aiming at the bottleneck problem of PDT, so that the distribution of the photosensitizer ALA in skin squamous carcinoma tissues/non-cancerous tissues is changed, the sensitivity of the photosensitizer is improved through RNA interference, the PDT treatment index is improved, and the toxic and side effects caused by nonspecific absorption of the photosensitizer are reduced.

Description

Targeted anti-skin squamous cell carcinoma co-loaded super-deformable liposome and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a co-loaded super-deformable liposome for targeting anti-skin squamous cell carcinoma, and a preparation method and application thereof.

Background

Cutaneous squamous cell carcinoma (cSCC) is one of the most common and most harmful cutaneous malignancies of the human head, face and genitalia, accounting for approximately 20% of non-melanotic skin tumors, with squamous cell carcinoma of the head and neck being more common. Studies have shown that some patients with cSCC, which has a particular risk factor for disease, are prone to relapse, metastasis, or even death; such as lesion with diameter more than or equal to 2cm, lesion located in ear or red lip, facial mask region, hand, foot, genitalia or embryo fusion part, lesion with thickness more than 2mm and low tissue differentiation or invasion to subcutaneous tissue (peripheral nerve, blood vessel or lymph tissue). The prognosis of lesions with involvement of known nerves or involvement of nerves greater than 0.1mm in diameter is poor. Tumor cells from immunocompromised cSCC patients show greater invasiveness, greatly increased rates of local and distant metastasis, and higher recurrence and mortality rates than immunocompromised patients. Patients with excessive exposure to uv radiation, radiation therapy or chronic skin lesions (wounds, ulcers, burns) have a significantly increased risk of developing invasive and metastatic sccc. Photodynamic Therapy (PDT) is a minimally invasive, scar-free Therapy with selective cytotoxicity, which is based on the principle that a photosensitizer is efficiently introduced into tumor cells, and a light source with a specific wavelength is given to irradiate a focal region, so that the photosensitizer absorbed by tissues is excited to generate active oxygen to cause tumor cell death, microvascular injury, local immunity induction and other reactions. PDT treatment of cSCC prior to or after surgery has become increasingly established, with the most commonly used Photosensitizers (PS) being 5-aminolevulinic acid (ALA) and Methyl aminolevulinic acid (MAL). However, ALA-PDT treatment suffers from the bottlenecks of weak penetration depth of the photodynamic light source, low ALA skin permeability, skin toxicity due to nonspecific absorption of photosensitizers, and reduced photodynamic treatment effect due to hypoxia at tumor sites.

HIF-1 α and its target gene VEGF have been shown to be highly expressed in skin malignancies such as squamous cell carcinoma of skin, basal cell carcinoma, etc., both of which exert important effects in cell tolerance to hypoxia, promotion of tumor cell proliferation, invasion, vascularization, metastasis and multidrug resistance, PDT has been demonstrated to promote expression of HIF-1 α and VEGF in paraneoplastic tissues while PDT is effective in treating tumors, whereas HIF-1 α can regulate tumor angiogenesis in PDT by VEGF-dependent and VEGF-independent pathways, i.e., HIF-1 α has a certain effect in protecting against photodynamic therapy of tumors, thus HIF- α can be used as a therapeutic agent for local HIF-1 interference, thus reducing tumor proliferation and reducing tumor cell sensitivity by targeted RNA therapy, and thus reducing tumor proliferation and improving tumor cell sensitivity by PDT.

With the continuous research of nano science, the application of nano-carriers in gene transportation, drug loading and other aspects is rapidly developed, such as liposomes, polymer nanoparticles, nano-micelles, inorganic gold nanoparticles, magnetic nanoparticles and the like, wherein the super-deformable liposomes (ProTS) are particularly suitable for skin drug delivery, so that the skin permeation of the loaded drugs can be promoted, the surface modification of the super-deformable liposomes can be easily realized, the nano-carriers become the most common carriers for constructing co-loaded targeted drug delivery systems of chemotherapeutic drugs, gene drugs and the like, and the ProTS are more easily absorbed by HNSCC through the skin compared with other nano-carriers.

Signal transducers and transcriptional activators 3 (STAT 3) are a family of proteins that exist in the cytoplasm and can be transferred into the nucleus to bind to DNA after activation, and have dual functions of signal transduction and transcriptional regulation. Activation of STAT3 initiates transcription of multiple cancer-associated genes, promotes cell proliferation, inhibits apoptosis, promotes angiogenesis, promotes migration and invasion of cells, and thus STAT3 is considered to be an oncogene. Recent research shows that the expression of phosphorylated STAT3 in cSCC is more than that of normal skin, phosphorylated STAT3 can induce the over-expression of cyclinD1, thereby promoting the tumor cells to maintain a high proliferation state, ALA-PDT can reduce the expression of STAT3, and therefore, the selective inhibition of STAT3 expression is expected to become a new strategy for cSCC treatment.

rS3-PA is a recombinant specific peptide aptamer of STAT3, can penetrate skin by local application, enter skin cells, mediate translocation and endocytosis through a Protein Transduction Domain (PTD) with nine arginine with positive electricity on the surface of a cell membrane to enter cytoplasm aggregation (independent of an endosome and a lysosome pathway), reduce the activation level of STAT3 in tumor cells and degrade phosphorylated STAT 3. Therefore, the peptide aptamer rS3-PA is used as a targeting functional group for transdermal drug delivery of the drug-loaded nano deformed liposome, so that activation of STAT3 can be inhibited while carrier delivery is promoted. Compared with antibodies, the aptamer has the advantages of small molecular weight, easiness in chemical synthesis and modification, good stability, small immunogenicity and the like. Peptide aptamers are short peptides consisting of 12-20 amino acids, can be combined with a specific functional region of a target protein, and are widely used for peptide identification.

Therefore, theoretically, rS3-PA modified super-deformable liposomes (ProTS) can be used for constructing a nano targeting drug delivery system loaded with ALA and HIF-1 α siRNA together [ rS3-PA-ProTS/(ALA/HIF-1 α siRNA) ], so that the distribution of a photosensitizer ALA in skin squamous carcinoma tissues/non-cancer tissues is changed, the sensitivity of the photosensitizer is improved, the PDT treatment index is improved, and meanwhile, the toxic and side effects caused by nonspecific absorption of the photosensitizer are reduced.

Disclosure of Invention

The first purpose of the invention is to provide a co-loaded super-deformed liposome targeting anti-skin squamous cell carcinoma aiming at the defects in the prior art.

The second purpose of the present invention is to provide a method for preparing the co-loaded superproteoliposome as described above, aiming at the disadvantages of the prior art.

A third object of the present invention is to overcome the disadvantages of the prior art by providing a pharmaceutical use of the above-mentioned co-loaded hyper-deformable liposomes.

The fourth purpose of the invention is to provide a gel for treating skin squamous cell carcinoma, which aims at the defects in the prior art.

In order to achieve the first purpose, the invention adopts the technical scheme that:

a co-carried supermorphic liposome targeted against skin squamous cell carcinoma is a peptide aptamer rS3-PA modified supermorphic liposome, and consists of hydrogenated soybean phospholipid, sodium cholate, poloxamer and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, and the supermorphic liposome is co-carried with 5-aminolevulinic acid and hypoxia inducible factor 1- α siRNA.

As a preferred embodiment of the present invention, the weight ratio of the hydrogenated soybean phospholipid, sodium cholate, poloxamer and distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 10:1:1: 0.1.

As a preferred embodiment of the invention, the sequence of the hypoxia inducible factor 1- α siRNA is CUGAUGACCAGCAACUUGA.

As a preferred embodiment of the present invention, the amino acid sequence of the peptide aptamer rS3-PA is Val-Arg-His-Ser-Ala-Leu-His-Met-Ala-Val-Gly-Pro-Leu-Ser-Trp-Pro-Ala-Arg-Val-Ser.

As a preferred embodiment of the invention, the preparation method of the co-loaded super-deformable liposome targeting the anti-skin squamous cell carcinoma comprises the following steps:

(1) distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-N-hydroxysuccinimide reacts with peptide aptamer rS3-PA to prepare DSPE-PEG₂₀₀₀-rS3-PA；

(2) Mixing hydrogenated soybean phospholipid, sodium cholate, poloxamer, distearoyl phosphatidyl ethanolamine-polyEthylene glycol 2000 and DSPE-PEG₂₀₀₀-rS3-PA is dissolved in isopropanol, rotary evaporation is carried out to form a lipid film, 0.9% sodium chloride solution dissolved with ALA is added until the film is completely hydrated, and liposomes are extruded;

(3) dissolving hypoxia inducible factor 1- α siRNA in diethyl pyrocarbonate water, and mixing with liposome.

As a preferred embodiment of the present invention, the molar ratio of the peptide aptamer rS3-PA to distearoylphosphatidylethanolamine-polyethylene glycol 2000-N-hydroxysuccinimide is 1: 4.

As a preferred embodiment of the present invention, the step 2 employs a polycarbonate membrane with a pore size of 220nm-80nm for high pressure filtration to extrude the liposome.

As a preferred embodiment of the invention, the mass ratio of ALA to hydrogenated soybean phospholipids in the step 2 is 1: 6-10.

As a preferred embodiment of the present invention, the final concentration of hypoxia inducible factor 1- α siRNA in step 3 is 0.5 mmol/L.

In order to achieve the second object, the invention adopts the technical scheme that:

the preparation method of the co-loaded super-deformed liposome comprises the following steps:

(4) The molar ratio of the peptide aptamer rS3-PA to the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-N-hydroxysuccinimide is 1:4, the step 2 is to filter and extrude liposome by adopting a polycarbonate membrane with the pore diameter of 220nm-80nm at high pressure, the mass ratio of ALA to hydrogenated soybean phospholipid in the step 2 is 1:6-10, the final concentration of hypoxia inducible factor 1- α siRNA in the step 3 is 0.5mmol/L, and the weight ratio of the hydrogenated soybean phospholipid, sodium cholate, poloxamer and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is 10:1:1: 0.1.

In order to achieve the third object, the invention adopts the technical scheme that:

use of a co-loaded superproteoliposome according to any of the above in the manufacture of a medicament for the treatment of cutaneous squamous cell carcinoma.

In order to achieve the fourth object, the invention adopts the technical scheme that:

a gel for treating skin squamous cell carcinoma, which is prepared from the co-carried super deformed liposome and a conventional gel matrix.

The conventional gel matrix may be 2% carboxyvinyl polymer (CP-940), and preferably, the concentration of the co-supported super-proteolipid in the gel is 0.001-10 g/ml.

Aiming at the bottleneck problem existing in PDT, the invention firstly constructs the peptide aptamer rS3-PA mediated photosensitizer ALA and HIF-1 α siRNA combined co-carried super-deformable liposome, thereby not only changing the distribution of the photosensitizer ALA in skin squamous carcinoma tissues/non-cancer tissues, but also improving the sensitivity of the photosensitizer through RNA interference, realizing the improvement of PDT treatment index and simultaneously reducing the toxic and side effects caused by non-specific absorption of the photosensitizer.

Aiming at the characteristic of difficult carrier selection in the combined application of the photosensitizer and gene therapy, the super-deformable liposome modified by the aptamer is used as a co-carrier of the photosensitizer and the siRNA, so that the stability of ALA can be improved, the premature release of water-soluble siRNA can be avoided, and the skin permeation of ALA can be promoted.

According to the invention, rS3-PA is combined with ALA and HIF-1 α siRNA, and the result shows that rS3-PA has a synergistic effect with ALA and HIF-1 α siRNA, and the prepared super deformed liposome has a remarkably enhanced effect on resisting skin squamous cell carcinoma.

In the co-loading targeting super-deformed liposome, the activity maintenance of the peptide aptamer rS3-PA is the key of the manifestation of the biological properties of the nanoparticle. The modification of rS3-PA is realized through the amino reaction of DSPE-PEG2000-NHS and rS3-PA, and the structure of aptamer rS3-PA is not changed in the whole process, so that the biological activity of the aptamer rS3-PA can be maintained.

Drawings

FIG. 1 shows the uptake of different intervention drugs by A431 cells.

FIG. 2 is a graph of ALA concentration versus time in different dialysates.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1 preparation and Performance characterization of Co-loaded Superproteoliposome [ rS3-PA-ProTS/(ALA/HIF-1 α siRNA) ] targeting anti-cutaneous squamous cell carcinoma

Firstly, rS3-PA is used for modifying super deformed liposomes (protS) to construct a nano targeting drug delivery system carrying ALA and HIF-1 α siRNA together [ rS3-PA-protS/(ALA/HIF-1 α siRNA) ], and the preparation method is as follows:

(1) synthesis of HIF-1 α siRNA human HIF-1 α siRNA (sequence: 5'-CUGAUGACCAGCAACUUGAtt-3') and control HIF-1 α siRNA were synthesized according to applicants ' earlier published literature [ Chen ZJ, et al. int. J. N. minor, 2016; 11: 991-.

(2) According to the literature [ Borghouts C, et al.jakstat, 2012; 1(1) 44-54 ] rS3-PA is synthesized, and the sequence is: N-VRH SAL HMA VGP LSW PAR VS-C.

(3) Synthesis of DSPE-modified rS3-PA: by distearoylphosphatidylethanolamine-polyethylene glycol₂₀₀₀-N-hydroxysuccinimide (DSPE-PEG)₂₀₀₀-NHS) with the amino group of rS3-PA to produce a stable amide bond to obtain DSPE-PEG₂₀₀₀-rS 3-PA. The specific scheme is as follows: precisely weighing appropriate amount of rS3-PA and DSPE-PEG₂₀₀₀-NHS (molar ratio rS3-PA: DSPE-PEG₂₀₀₀-NHS ═ 1:4), dissolved in DMF respectively, the two were mixed and stirred at room temperature for 24h to mix well; and adding triethylamine to adjust the solution to be alkalescent (pH is 7.0-8.0), continuously reacting for 36h at room temperature, tracking the reaction by adopting thin-layer chromatography until the amount of rS3-PA is not reduced any more, dialyzing the reaction solution by using purified water in a dialysis bag, and freeze-drying to obtain the compound.

(4) Preparing target super deformed liposome rS3-PA-ProTS/(ALA/HIF-1 α siRNA), precisely weighing HSPC, sodium cholate, F-68 and DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀0-rS3-PA (mass ratio of 10:1:1:0.1:0.1), putting into a dry clean rotary evaporation bottle, adding isopropanol until the isopropanol is completely dissolved, forming a lipid film by rotary evaporation under the vacuum condition, then adding 0.9% sodium chloride solution dissolved with ALA (the mass ratio of ALA to HSPC is 1:10 or 1:6) until the film is completely hydrated, carrying out vortex oscillation to uniformly distribute the lipid film, sucking out all liposomes, placing in an ice water mixture for ultrasonic treatment, respectively adopting 220nm and 80nm polycarbonate films for high-pressure filtration to extrude the liposomes, dissolving HIF-1 α siRNA in diethyl pyrocarbonate (DEPC) water, and uniformly mixing with the liposome solution under the aseptic condition to ensure that the final concentration of the siRNA is 0.5 mmol/L.

Second, characterization of Properties

(1) The result shows that in rS3-PA-ProTS/(ALA/HIF-1 α siRNA), the entrapment rate of siRNA is 91.9%, the entrapment rate of ALA is 64.96% (the mass ratio of ALA to HSPC is 1:6), and 74.17% (the mass ratio of ALA to HSPC is 1:10), and the result shows that the prepared super-deformable liposome has good entrapment rate.

(2) Measuring ALA content by adopting a fluorescamine pre-column derivatization HPLC-fluorescence method under the chromatographic conditions that an Inertsil ODS-3C18 column (4.6mm × 250mm, 5m) is adopted as a chromatographic column, acetonitrile-0.1% trifluoroacetic acid solution (30: 70) is adopted as a mobile phase, the excitation wavelength is 398nm, the emission wavelength is 480nm, the column temperature is 40 ℃, the flow rate is 1.5ml/min, and the sample injection amount is 20 mu l.

Three, a synergistic action mechanism among different drugs in the co-loaded targeting super-deformed liposome

(1) Preparing target super deforming liposome rS3-PA-ProTS, rS3-PA-ProTS/ALA, rS3-PA-ProTS/HIF-1 α siRNA, rS3-PA-ProTS/(ALA/HIF-1 α siRNA), 4 kinds of the preparation method comprises the following steps:

① rS3-PA-ProTS, precisely weighing HSPC, sodium cholate, F-68 and DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀0-rS3-PA (mass ratio 10:1:1:0.1:0.1) is put into a dry clean rotary evaporation bottle and added with isopropanol until all the isopropanol is dissolved. Under vacuum condition, rotary evaporation forms lipid film. Then 0.9% sodium chloride solution was added until the film hydration was complete. Vortex shaking made the lipid film evenly distributed. All liposomes were aspirated and sonicated in an ice water mixture. The liposome is extruded by high pressure filtration with polycarbonate membranes of 220nm and 80nm respectively.

② rS3-PA-ProTS/ALA is prepared by precisely weighing HSPC, sodium cholate, F-68, and DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀0-rS3-PA (mass ratio 10:1:1:0.1:0.1) is put into a dry clean rotary evaporation bottle and added with isopropanol until all the isopropanol is dissolved. Under vacuum condition, rotary evaporation forms lipid film. Then 0.9% sodium chloride solution dissolved with ALA (mass ratio of ALA to HSPC is 1:10) is added until the hydration of the film is complete. Vortex shaking made the lipid film evenly distributed. All liposomes were aspirated and sonicated in an ice water mixture. The liposome is extruded by high pressure filtration with polycarbonate membranes of 220nm and 80nm respectively.

③ rS3-PA-ProTS/HIF-1 α siRNA is prepared by precisely weighing HSPC, sodium cholate, F-68, DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀0-rS3-PA (mass ratio 10:1:1:0.1:0.1) is put into a dry clean rotary evaporation bottle and added with isopropanol until all the isopropanol is dissolved. Under vacuum condition, rotary evaporation forms lipid film. Then 0.9% chlorine was addedDissolving sodium solution until the film is completely hydrated, carrying out vortex oscillation to uniformly distribute lipid membranes, sucking out all liposomes, placing in an ice water mixture for ultrasonic treatment, respectively adopting 220nm and 80nm polycarbonate membranes for high-pressure filtration to extrude the liposomes, dissolving HIF-1 α siRNA in diethyl pyrocarbonate (DEPC) water, and uniformly mixing with the liposome solution under the aseptic condition to ensure that the final concentration of the siRNA is 0.5 mmol/L.

④ rS3-PA-ProTS/(ALA/HIF-1 α siRNA) is prepared by precisely weighing HSPC, sodium cholate, F-68, and DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀0-rS3-PA (mass ratio of 10:1:1:0.1:0.1), putting into a dry clean rotary evaporation bottle, adding isopropanol until the isopropanol is completely dissolved, forming a lipid film by rotary evaporation under the vacuum condition, then adding 0.9% sodium chloride solution dissolved with ALA (the mass ratio of ALA to HSPC is 1:10 or 1:6) until the film is completely hydrated, carrying out vortex oscillation to uniformly distribute the lipid film, sucking out all liposomes, placing in an ice water mixture for ultrasonic treatment, respectively adopting 220nm and 80nm polycarbonate films for high-pressure filtration to extrude the liposomes, dissolving HIF-1 α siRNA in diethyl pyrocarbonate (DEPC) water, and uniformly mixing with the liposome solution under the aseptic condition to ensure that the final concentration of the siRNA is 0.5 mmol/L.

The super modified liposome is added into 2 percent carboxyvinyl polymer (CP-940) to prepare gel for subsequent experiments.

(2) Cell culture: a431 and SCL-1 human epidermal squamous carcinoma cell (purchased from cell bank of Chinese academy of sciences) is cultured in RPMI1640 medium containing 10% FBS, 100U/ml penicillin and 100. mu.g/ml streptomycin at 37.0 deg.C and 5% CO₂Culturing in an incubator, observing cell morphology and growth every day, and replacing culture solution every 2 days.

(3) Cell proliferation inhibition experiment of cells to be in logarithmic growth phase (3 × 10)⁴/cm²100. mu.l) were inoculated into a 96-well plate, and when about 90% of the cells were fused, the serum-containing culture solution was aspirated and washed twice with PBS. Adding non-medicated serum-free culture medium 100 μ l in the control group, and culturing in dark place; adding the above 4 target super-deformable liposomes containing different drugs and 100 μ l of serum-free culture medium into other groups, wrapping with foil paper, culturing in dark for 24 hr, and administering ALAIrradiation with 635nm Red light (8J/cm)²，8.6mW/cm²). After irradiation, each group was cultured for 24 hours, and MTT was used to measure the cell growth inhibition rate.

TABLE 1 inhibition of cell growth by different treatments

The results show that the rS3-PA, ALA and HIF-1 α siRNA are combined to have the effect of synergistically inhibiting the proliferation of tumor cells.

(4) Apoptosis test comprises treating cultured cells according to the above 4 different intervention measures, culturing for 24 hr, collecting cells, rinsing with precooled PBS buffer solution, and performing cell apoptosis test according to 1 × 10⁶Resuspending the cells at a density of/ml, then placing 100 μ l of the cells in a flow tube, adding 5 μ l each of Annexin V-FITC and propidium iodide, incubating for 15min in the dark, then detecting 10000 cells by flow cytometry, and finally analyzing the result of apoptosis by Cell Quest software.

TABLE 2 apoptosis rates of different treatment groups

The results show that the rS3-PA, ALA and HIF-1 α siRNA are combined to have the effect of synergistically promoting the apoptosis of tumor cells.

Targeted effect evaluation of four-loading-target super-deformed liposome

In vitro targeting is to take A431 cells in a logarithmic growth phase, inoculate the cells to a 12-well plate, add a co-load targeting hyper-deformable liposome marked by 6-FAM, investigate the uptake conditions of the cells to ProTS/(ALA/HIF-1 α siRNA) and rS3-PA-ProTS/(ALA/HIF-1 α siRNA) through a fluorescence microscope, and evaluate the in vitro targeting.

Fifthly, evaluation of in-vitro skin permeability of co-loaded targeting super-deformed liposome

(1) Isolated skin permeation isolated from BALL/C nude mice dorsal skin ex vivo, subcutaneous adipose tissue was removed, and the isolated skin permeation was evaluated by using a modified Franz diffusion cell assay, wherein ProTS/(ALA/HIF-1 α siRNA) gel and rS3-PA-ProTS/(ALA/HIF-1 α siRNA) gel (containing ALA 50mg/g and siRNA 0.5. mu. mol/g) were administered to the supply chamber, and the receiving solution was collected at 0.5, 1, 2, 4, 6, and 8 hours after administration, centrifuged, and assayed for ALA content by HPLC method.

TABLE 3 ALA cumulative permeation at different times (μ g cm)^-2)(n＝6)

The results show that the hyper-deformable liposome can promote ALA transdermal, and the rS3-PA and the hyper-deformable liposome are combined to further promote ALA transdermal effect.

Sixth, research on in vivo skin permeability of co-loaded targeting super-deformed liposome

(1) Constructing a local microdialysis sampling system for the transplanted tumor: a431 cell strain is used for constructing a subcutaneous transplanted tumor model of a nude mouse with human skin squamous cell carcinoma, a local microdialysis sampling system of the transplanted tumor is constructed by adopting a percutaneous microdialysis technology, and a microdialysis solution sample is dynamically collected in real time.

(2) In the body skin penetration characteristic study, ProTS/(ALA/HIF-1 α siRNA) gel and rS3-PA-ProTS/(ALA/HIF-1 α siRNA) gel (containing ALA 50mg/g and siRNA 0.5 mu mol/g) are respectively administrated to the skin part of the sampling system, one sample is collected every 60 minutes after the administration, the sample is collected for 8 hours, the ALA content is determined by an HPLC method, and the characteristics of the targeted super deformed liposome gel in the body skin penetration are clarified by matching the local pharmacokinetic data of tumors.

TABLE 4 local ALA pharmacokinetic parameters after transdermal administration of different gels

Seven, in vivo drug effect of co-loaded nano-targeting super-deformation liposome

① model construction of nude mouse graft tumor of skin squamous cell carcinoma, skin squamous cell carcinoma A431 cell is added at concentration of 1 × 10⁶0.2 ml/mouse is planted under the skin of a nude mouse (BALB/c-nu/nu) after being mixed with matrigel in a ratio of 1:1 to establish a model of subcutaneous transplantation tumor of the nude mouse with the human skin squamous cell carcinoma.

② evaluation of drug effect of co-loading targeting hypermorphic liposome, constructed nude mice with human skin squamous cell carcinoma are transplanted with tumor model mice, pathological biopsy is confirmed to be tumor-bearing mice of SCC, the mice are randomly divided into 2 groups, 10 mice in each group are respectively and locally administered with equal amount of rS3-PA-ProTS/(ALA/HIF-1 α siRNA) gel and pure hypermorphic liposome ProTS without drugs on the skin, and simultaneously, the light source is used for irradiation, and the tumor inhibition effect of rS3-PA-ProTS/(ALA/HIF-1 α siRNA) is observed.

Evaluation criteria: observing and recording the tumor changes of the tumor-bearing mice before and two weeks after each treatment, and measuring the maximum diameter (a) and the minimum diameter (b) of the tumor of the mice by using a vernier caliper, wherein the tumor volume (V) of the mice is ab ²2, maximum tumor volume greater than 500mm³The experiment is terminated, the mice are anesthetized with 7% chloral hydrate, cervical vertebrae are removed and killed, and the number of tumor bodies with the diameter larger than 1mm is counted manually. And (3) complete alleviation: tumor mass regresses, only pigmentation or hypopigmentation remains, tissue biopsy is normal skin structure; partial mitigation: the tumor body is reduced by more than 50 percent; no reaction: the tumor volume is reduced by less than 50 percent or the skin damage is not obviously changed.

The results are shown in the table below, wherein the number of tumor bodies and the average tumor volume of the two groups before treatment are not statistically different, wherein the rS3-PA-ProTS/(ALA/HIF-1 α siRNA) gel group has 57 tumor bodies before treatment, 48 tumor bodies are completely relieved after 2 weeks of treatment, the complete remission rate is 84.2% (48/57), the partial remission rate is 8.8% (5/57), and the inefficiency is 7.0% (4/57). the pure hyperprote liposome group has 3.4% (2/58) and 96.6% (56/58) of partial remission rate.

TABLE 5 rS3-PA-ProTS/(ALA/HIF-1 α siRNA) gel and drug-free pure superproteoliposome ProTS tumor-inhibiting effect

Aiming at the bottleneck problem existing in PDT, the invention constructs the peptide aptamer rS3-PA mediated photosensitizer ALA and HIF-1 α siRNA combined co-carried super-deformable liposome, thereby not only changing the distribution of the photosensitizer ALA in skin squamous carcinoma tissues/non-cancer tissues, but also improving the sensitivity of the photosensitizer through RNA interference, realizing the improvement of PDT treatment index, and simultaneously reducing the toxic and side effects caused by nonspecific absorption of the photosensitizer.

Example 2 preparation of Co-loaded Superproteoliposome [ rS3-PA-ProTS/(ALA/HIF-1 α siRNA) ] targeting anti-cutaneous squamous cell carcinoma

Synthesis of DSPE-modified rS3-PA: precisely weighing appropriate amount of rS3-PA and DSPE-PEG₂₀₀₀-NHS (molar ratio rS3-PA: DSPE-PEG₂₀₀₀-NHS ═ 1:2), dissolved in DMF respectively, the two were mixed and stirred at room temperature for 24h to mix well; and adding triethylamine to adjust the solution to be alkalescent (pH is 7.0-8.0), continuously reacting for 36h at room temperature, tracking the reaction by adopting thin-layer chromatography until the amount of rS3-PA is not reduced any more, dialyzing the reaction solution by using purified water in a dialysis bag, and freeze-drying to obtain the compound.

Preparation of rS3-PA-ProTS/(ALA/HIF-1 α siRNA) by precisely weighing HSPC, sodium cholate, F-68 and DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀0-rS3-PA (mass ratio is 4:4:4:0.1:0.1), putting into a dry clean rotary evaporation bottle, adding isopropanol until the isopropanol is completely dissolved, forming a lipid film by rotary evaporation under the vacuum condition, then adding 0.9% sodium chloride solution dissolved with ALA (the mass ratio of ALA to HSPC is 1:2) until the film is completely hydrated, carrying out vortex oscillation to ensure that the lipid film is uniformly distributed, sucking out all the liposomes, putting into an ice water mixture for ultrasonic treatment, respectively adopting 220nm and 80nm polycarbonate films for high-pressure filtration to extrude the liposomes, dissolving HIF-1 α siRNA into diethyl pyrocarbonate (DEPC) water, uniformly mixing with the liposome solution under the aseptic condition to ensure that the final concentration of the siRNA is 0.2mmol/L, adding the super-deformed liposome into 2% carbopol (CP-940) to prepare the gel.

Example 3 preparation of Co-loaded Superproteoliposome [ rS3-PA-ProTS/(ALA/HIF-1 α siRNA) ] targeting anti-cutaneous squamous cell carcinoma

Synthesis of DSPE-modified rS3-PA: precisely weighing appropriate amount of rS3-PA and DSPE-PEG₂₀₀₀-NHS (molar ratio rS3-PA: DSPE-PEG₂₀₀₀-NHS ═ 1:6), dissolved in DMF respectively, the two were mixed and stirred at room temperature for 24h to mix well; and adding triethylamine to adjust the solution to be alkalescent (pH is 7.0-8.0), continuously reacting for 36h at room temperature, tracking the reaction by adopting thin-layer chromatography until the amount of rS3-PA is not reduced any more, dialyzing the reaction solution by using purified water in a dialysis bag, and freeze-drying to obtain the compound.

Preparation of rS3-PA-ProTS/(ALA/HIF-1 α siRNA) by precisely weighing HSPC, sodium cholate, F-68 and DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀0-rS3-PA (mass ratio 2:6:6:0.1:0.1), putting into a dry clean rotary evaporation bottle, adding isopropanol to completely dissolve, forming a lipid film by rotary evaporation under the vacuum condition, then adding 0.9% sodium chloride solution dissolved with ALA (the mass ratio of ALA to HSPC is 1:4) until the film is completely hydrated, carrying out vortex oscillation to ensure that the lipid film is uniformly distributed, sucking out all the liposomes, putting into an ice water mixture for ultrasonic treatment, respectively adopting 220nm and 80nm polycarbonate films for high-pressure filtration to extrude the liposomes, dissolving HIF-1 α siRNA into diethyl pyrocarbonate (DEPC) water, uniformly mixing with the liposome solution under the aseptic condition to ensure that the final concentration of the siRNA is 0.1mmol/L, adding 2% carbopol into the super deformation liposome (CP-940)Chinese medicinal gel

Example 4 preparation of Co-loaded Superproteoliposome [ rS3-PA-ProTS/(ALA/HIF-1 α siRNA) ] targeting anti-cutaneous squamous cell carcinoma

Synthesis of DSPE-modified rS3-PA: precisely weighing appropriate amount of rS3-PA and DSPE-PEG₂₀₀₀-NHS (molar ratio rS3-PA: DSPE-PEG₂₀₀₀-NHS ═ 1:3), respectively dissolved in DMF, the two were mixed and stirred at room temperature for 24h to mix well; and adding triethylamine to adjust the solution to be alkalescent (pH is 7.0-8.0), continuously reacting for 36h at room temperature, tracking the reaction by adopting thin-layer chromatography until the amount of rS3-PA is not reduced any more, dialyzing the reaction solution by using purified water in a dialysis bag, and freeze-drying to obtain the compound.

Preparation of rS3-PA-ProTS/(ALA/HIF-1 α siRNA) by precisely weighing HSPC, sodium cholate, F-68 and DSPE-PEG₂₀₀₀、DSPE-PEG₂₀₀0-rS3-PA (mass ratio 5:4:3:0.1:0.1), putting into a dry clean rotary evaporation bottle, adding isopropanol to completely dissolve, forming a lipid film by rotary evaporation under the vacuum condition, then adding 0.9% sodium chloride solution dissolved with ALA (the mass ratio of ALA to HSPC is 1:12) until the film is completely hydrated, carrying out vortex oscillation to ensure that the lipid film is uniformly distributed, sucking out all the liposomes, putting into an ice water mixture for ultrasonic treatment, respectively adopting 220nm and 80nm polycarbonate films for high-pressure filtration to extrude the liposomes, dissolving HIF-1 α siRNA into diethyl pyrocarbonate (DEPC) water, uniformly mixing with the liposome solution under the aseptic condition to ensure that the final concentration of the siRNA is 0.8mmol/L, adding the super-deformed liposome into 2% carbopol (CP-940) to prepare a gel

Evaluation of the efficacy of the superproteoliposomes prepared in the different examples:

the established nude mice bearing tumor model mouse with human skin squamous cell carcinoma, tumor bearing mice with SCC confirmed by pathological biopsy, were randomly divided into 4 groups, 10 mice in each group, and the rS3-PA-ProTS/(ALA/HIF-1 α siRNA) gel prepared in example 1-4 was administered in equal amount to the skin topically while irradiating with light source.

Evaluation criteria: the tumor change of the tumor-bearing mice before and after two weeks of each treatment is observed and recordedThe maximum diameter (a) and the minimum diameter (b) of a mouse tumor, and the tumor volume (V) of the mouse is ab ²2, maximum tumor volume greater than 500mm³The experiment is terminated in time, a 7% chloral hydrate is used for anesthetizing a mouse, cervical vertebra is removed for sacrifice, the number of tumor bodies with the diameter larger than 1mm is counted manually, the tumor bodies are completely relieved, only pigmentation or hypopigmentation is remained, tissue biopsy is a normal skin structure, partial relief is that the tumor bodies are reduced by more than 50%, no response is made, the tumor bodies are reduced by less than 50% or no obvious change is caused, and the results are shown in the following table, and the rS3-PA-ProTS/(ALA/HIF-1 α siRNA) prepared in example 1 has the optimal tumor inhibition effect.

TABLE 6 tumor suppression Effect of Targeted Superproteoliposomes of the different examples

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Hospital for skin diseases of Shanghai city

<120> co-carried super deformed liposome for targeting anti-skin squamous cell carcinoma, preparation method and application thereof

<130>/

<160>2

<170>PatentIn version 3.3

<210>1

<211>19

<212>RNA

<213> Artificial sequence

<400>1

cugaugacca gcaacuuga 19

<210>2

<211>20

<212>PRT

<213> Artificial sequence

<400>2

Val Arg His Ser Ala Leu His Met Ala Val Gly Pro Leu Ser Trp Pro

1 5 10 15

Ala Arg Val Ser

20

Claims

1. The co-carried super deformed liposome targeted against skin squamous cell carcinoma is characterized in that the super deformed liposome is a super deformed liposome modified by a peptide aptamer rS3-PA, the super deformed liposome is composed of hydrogenated soybean phospholipid, sodium cholate, poloxamer and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, the super deformed liposome is co-carried with 5-aminolevulinic acid and hypoxia inducible factor 1- α siRNA, the weight ratio of the hydrogenated soybean phospholipid, the sodium cholate, the poloxamer and the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is 10:1:1:0.1, the sequence of the hypoxia inducible factor 1- α siRNA is CUGAUGACCAGCAACUUGA, the amino acid sequence of the peptide aptamer rS3-PA is Val-Arg-His-Ser-Ala-Leu-His-Met-Ala-Gly-Pro-Leu-Ser-Trp-Pro-Ala-Arg-Val-Ser, and the molar ratio of the peptide aptamer rS3-PA to the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-N-hydroxysuccinimide is 1: 4.

2. The co-entrapped superproteusliposome targeted to anti-cutaneous squamous cell carcinoma according to claim 1, wherein said co-entrapped superproteusliposome targeted to anti-cutaneous squamous cell carcinoma is prepared by the following method:

(1) distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-N-hydroxysuccinimide reacts with the peptide aptamer rS3-PA to prepare DSPE-PEG2000-rS 3-PA;

(2) dissolving hydrogenated soybean phospholipid, sodium cholate, poloxamer, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and DSPE-PEG2000-rS3-PA in isopropanol, performing rotary evaporation to form a lipid film, adding 0.9% sodium chloride solution dissolved with ALA until the film is completely hydrated, and extruding liposome;

(3) dissolving hypoxia inducible factor 1- α siRNA in diethyl pyrocarbonate water, and mixing with liposome to obtain the final product.

3. The co-loaded superdeformable liposome targeted against cutaneous squamous cell carcinoma according to claim 2, wherein the liposome is extruded in step 2 by high pressure filtration using a polycarbonate membrane with pore size of 220nm-80nm, the mass ratio of ALA to hydrogenated soybean phospholipid is 1:6-10, and the final concentration of hypoxia inducible factor 1- α siRNA in step 3 is 0.5 mmol/L.

4. Use of the co-loaded superproteoliposome according to any of claims 1-3 for the preparation of a medicament for the treatment of cutaneous squamous cell carcinoma.

5. A gel for the treatment of cutaneous squamous cell carcinoma, said gel being prepared from the co-supported superproteoliposome of any of claims 1-3 and a conventional gel matrix.