CN113185574A

CN113185574A - Targeting cell-penetrating peptide for enhancing sensitivity of cervical cancer radiotherapy, modified nanoparticle compound and pharmaceutical application

Info

Publication number: CN113185574A
Application number: CN202110476144.4A
Authority: CN
Inventors: 赵艳; 张婷婷; 段钊; 师蕊婷
Original assignee: Second Affiliated Hospital Of College Of Medicine Of Xi'an Jiaotong University
Current assignee: Second Affiliated Hospital Of College Of Medicine Of Xi'an Jiaotong University
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-07-30

Abstract

The invention discloses a targeted penetrating peptide, modified nanoparticle complex and pharmaceutical application for enhancing the radiotherapy sensitivity of cervical cancer. The targeted penetrating peptide can enter cervical cancer with specificity and high efficiency, and by regulating the PI3K/AKT pathway and acting as a "molecular sponge" of microRNA, thereby improving the sensitivity of radiotherapy and reducing the side effects of radiotherapy. The present invention modifies PEI with small molecular disulfide bonds (-SS-), so that the toxicity of PEI can be greatly reduced; at the same time, the colloidal stability of the PEI-nucleic acid complex is also improved, and its circulation time in the body is prolonged; The tumor targeting polypeptide R11 modifies the PEI-nucleic acid complex, which solves the targeting problem of nanoparticles in gene therapy research.

Description

Targeting cell-penetrating peptide for enhancing sensitivity of cervical cancer radiotherapy, modified nanoparticle compound and pharmaceutical application

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a targeting cell-penetrating peptide for enhancing the sensitivity of cervical cancer radiotherapy, a modified nanoparticle compound and pharmaceutical application.

Background

Cervical cancer is one of the common malignant tumors in gynecology, the mortality rate is second to breast cancer, and the health of women is seriously threatened. The comprehensive treatment combining operation with radiotherapy and chemotherapy is the main means for treating cervical cancer at present, and the curative effect is internationally agreed. However, in cervical cancer patients undergoing radiation therapy, there is still about 13% of patients with local recurrence. This is due in large part to the development of radiotherapeutic tolerance in tumors, and to the fact that the molecular mechanisms of radiotherapeutic tolerance are currently unclear, it has hitherto been difficult to accurately predict the risk of radiotherapeutic tolerance prior to patient radiotherapeutic treatment; on the other hand, the high-dose radiotherapy not only can greatly damage the normal tissues of the pelvic region, but also can easily cause various complications; conversely, if the radiation dose is reduced, it is difficult to achieve effective treatment, and the tumor may recur. And radiosensitization is one of the important strategies to solve the above problems. However, the current sensitization methods including physical sensitization (such as heating, ultra-short microwave, etc.) and chemical sensitization (such as metridazon chemosensitizer) are difficult to achieve the best curative effect due to the lack of tumor targeting. Therefore, the development of a safe and efficient novel targeted gene therapy medicament for enhancing radiotherapy sensitivity, reducing radiotherapy dosage and reducing side effects becomes one of important treatment strategies for improving cervical cancer prognosis.

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that are more than 200 nucleotides in length. In recent years, researches show that the lncRNA expression abnormality is closely related to the occurrence and development of various tumors including cervical cancer, and the lncRNA expression abnormality promotes the proliferation, invasion and metastasis of tumor cells through changing the expression of tumor-related genes, influencing cell cycles, interfering the functions of miRNA and the like, so that the lncRNA is becoming a new target for tumor treatment. Growth arrest specific transcription factor 5 (GAS 5) is a lncRNA with cancer suppressor function, and its expression is down-regulated in many tumor cells. Researches show that exogenous high-expression GAS5 in cervical cancer cells can inhibit cell proliferation, induce apoptosis, cause cell cycle arrest, simultaneously reduce the invasion and migration capacity of cancer cells, increase the sensitivity of the cells to anticancer drugs cis-platinum, and inhibit the growth of transplanted tumors in animals. The cancer inhibition function of the GAS5 is closely related to the miR-196a, miR-205, miR-21 and the like of the GAS5 trap. As a molecular sponge, GAS5 can mediate the radiotherapy sensitivity of cervical cancer through miR-106 b. In conclusion, GAS5 is a novel therapeutic target for cervical cancer radiotherapy sensitization.

Cell-penetrating peptides (CPPs) are also of interest as a novel drug delivery tool. CPPs are small molecular polypeptides consisting of no more than 30 amino acid residues, have transmembrane transport capacity, can carry exogenous macromolecules into cells, and have no toxic effect on host cells within a certain concentration range. The effect of tumor targeting arginine polypeptides as drug delivery vehicles to transfer polypeptide fragments or micrornas in tumor cells has been studied.

The cell-penetrating peptide R9 is proved to be a novel targeted drug delivery system, can carry polypeptide fragments to target into tumor cells and play a biological role. However, the R9 complex system has the defect of poor stability, and the function of the system is influenced. The cationic polymer-Polyethyleneimine (PEI) is one of the most widely studied non-viral nano-carriers in recent years, a non-covalent polyelectrolyte complex can be formed by compressing DNA and RNA, and the small nano-particle size and surface positive charge of the complex are favorable for endocytosis of the complex. PEI is considered as an ideal drug delivery carrier because of the characteristics of higher transfection efficiency, low immune response, good biocompatibility and the like. However, the major bottlenecks affecting the application of PEI are its high cytotoxicity and lack of targeting.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a targeting cell-penetrating peptide for enhancing the sensitivity of cervical cancer radiotherapy, a modified nanoparticle compound and pharmaceutical application, wherein the targeting cell-penetrating peptide can enter the cervical cancer specifically and efficiently, and can improve the sensitivity of the radiotherapy and reduce the side effect of the radiotherapy by regulating a PI3K/AKT pathway and exerting the action of microRNA (ribonucleic acid) molecular sponges.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a targeting cell-penetrating peptide for enhancing cervical cancer radiotherapy sensitivity, which is R9 polypeptide, and the amino acid sequence of the targeting cell-penetrating peptide is shown in SED.ID.NO.1.

The invention also discloses a targeting cell-penetrating peptide nano drug delivery system for enhancing the sensitivity of cervical cancer radiotherapy, which is formed by coupling the R9 cell-penetrating peptide to polyethyleneimine PEI through a disulfide bond SS.

The invention also discloses a targeting cell-penetrating peptide nanoparticle compound for enhancing the sensitivity of cervical cancer radiotherapy, which is constructed by the nano drug delivery system R9-SS-PEI carrying lncRNA GAS 5; wherein: lncRNA GAS5 is lncRNA encoded by prostate cancer-associated locus lq 25.

The invention also discloses an application of the targeted cell-penetrating peptide nano drug delivery system or the targeted cell-penetrating peptide nano particle compound in preparation of drugs for treating cervical cancer.

Preferably, the medicament is a medicament with a targeting effect on cervical cancer.

Preferably, the medicine is a medicine for increasing the sensitivity of cervical cancer radiotherapy.

Preferably, the drug positively regulates the expression level of PI3K/Akt pathway, PTEN pathway or miR-106.

The invention discloses an application of the targeted cell-penetrating peptide nano drug delivery system or the targeted cell-penetrating peptide nano particle compound in preparation of a cervical cancer radiotherapy sensitizer.

Compared with the prior art, the invention has the following beneficial effects:

the invention uses small molecule disulfide bond (-SS-) to modify PEI, so that the toxicity of PEI is greatly reduced; meanwhile, the colloidal stability of the PEI-nucleic acid complex is improved, and the circulation time of the PEI-nucleic acid complex in vivo is prolonged; meanwhile, the PEI-nucleic acid complex is modified by the tumor targeting polypeptide R9, so that the targeting problem of the nanoparticles in gene therapy research is solved. Therefore, REG is expected to be developed as an effective means for enhancing the sensitivity of radiotherapy for cervical cancer.

Drawings

FIG. 1 is a schematic diagram of the synthesis of the R9-SSPEI-lncGAS nanoparticle complex of the present invention;

FIG. 2 shows the expression of lncgAS in cells after transfection of Hela cells with R9-SSPEI-lncgAS nanoparticle complexes at different time points;

FIG. 3 shows the cytotoxic effect of Hela, Siha under the effect of different mass ratios of R9-SSPEI/lncGAS nanoparticle complexes;

FIG. 4 is a graphical representation of the results of the establishment of the Hela/R and Siha/R cell lines of the chemotherapy-resistant cervical cancer cell lines; wherein A is Hela/R; b is Siha/R;

FIG. 5 is an image of the uptake of R9-SSPEI/lncgAS in tumor cells;

FIG. 6 shows the expression of lncGAS in a cell line resistant to and sensitive to cervical cancer radiotherapy and the effect on sensitivity to radiotherapy; wherein A is the expression difference B of LncGAS in cervical cancer cell lines (Hela, Siha) and radiotherapy-tolerant cell lines (Hela-R, Siha-R) thereof, and the expression difference B is the influence on the cell radiotherapy survival rate after the lncGAS is over-expressed in the radiotherapy-tolerant cell lines Hela-R; c is the influence of the overexpression of lnCGAS in a radiotherapy-tolerant cell line Siha-R on the survival rate of cell radiotherapy;

FIG. 7 shows the uptake of R9 in the cervical cancer in-situ model under the small animal imaging technique;

FIG. 8 is a graph showing that R9-SSPEI/lncgAS nanoparticles can inhibit tumor growth in the abdominal cavity; wherein A is the change of the tumor volume of the R9-SSPEI/lncgAS nano particles compared with the control group; and B is the change of tumor fluorescence under the action of the R9-SSPEI/lncGAS nano-particles and a control group.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the invention discloses a targeting cell-penetrating peptide for enhancing the sensitivity of cervical cancer radiotherapy, wherein the sequence of the targeting polypeptide is as follows: RRRRRRRRR are provided. The invention also discloses a tumor targeting nanoparticle for the radiotherapy sensitization of cervical cancer, and the nanoparticle can specifically enter the cervical cancer and play an anti-tumor effect in vivo and in vitro. In further research, the nano-particles can be specifically absorbed in a nude mouse in both an in-situ cervical cancer model and a metastasis model, and the effect of remarkably prolonging the survival time of a tumor-bearing mouse is achieved. However, the uptake rate of organs including ovary, heart, lung, liver, kidney and the like was low, and no cell damage effect was detected. In addition, PI3K/Akt pathway protein in the nanoparticle administration group of the tumor-bearing mice is remarkably reduced, and miR-106 has the effect of 'molecular sponge', which is very beneficial to enhancing radiotherapy sensitivity. In conclusion, the nanoparticle is a high-efficiency, specific and low-toxicity radiotherapy sensitizer.

Example 1 Synthesis of R9-SSPEI-lncgAS nanoparticle complexes

Referring to fig. 1, the specific synthesis process is as follows:

the synthesized lncGAS was dissolved in methanol (30ml) in a flask (100ml), and the sample was purified with liquid nitrogen and stored under vacuum for 10-20 min. Calculated amount of propylene sulfide (more than bpei1.2k 5 moles) was added by syringe. The solution was stirred at 60 ℃ for 24h, and the reaction mixture was evaporated to dryness under reduced pressure, then dissolved in methanol and precipitated twice in ice-cold diethyl ether. The mercaptan group content of the product was determined using an Ellman's method. BPEI-SH (0.5g) was dissolved in anhydrous dimethyl sulfoxide (50ml), and stirred at room temperature for 48 hours to oxidatively crosslink thiol groups. The product was purified by dialysis against d.i. water (mwco3500) and subjected to extensive lyophilization, the chemical structure of which was confirmed by proton Nuclear Magnetic Resonance (NMR). The extent of disulfide crosslinking was determined using the Ellman's method. BPEI-SS (12 equivalents) and MAL-PEG-NHS were dissolved in 50ml of anhydrous methanol and DMSO at a ratio of 1:5(v/v), respectively, stirred at room temperature for 4h, and then R9 polypeptide (1.2 equivalents and MAL-PEG-NHS) was added. The reaction mixture was stirred at room temperature for an additional 48 hours to afford BPEI-SS-PEG-R9.

To more accurately assess R9-mediated tumor targeting, methoxy polyethylene glycol conjugated BPEI-SS (BPEI-SS-MPEG) was used as a control. MPEG5 k (1g, 7mmol), NPC (400mg, 20mmol) and triethylamine (2.7ml, 7mmol) were dissolved in 50ml dichloromethane (MC) and reacted for 2h in an ice bath. After stirring the reaction mixture at room temperature for 16h, it was evaporated to dryness under reduced pressure, dissolved in MC and then precipitated twice in cold diethyl ether. 20ml of BPEI-SS (193mg, 0.16mmol) and NPC-activated MPEG (40mg, 6mmol) were dissolved in anhydrous methanol and dimethyl sulfoxide as solvents at a ratio of 1:3(v/v), stirred for 24h, and D.I. water (MWCO 10000) and freeze-dried water were purified in large quantities and proton nuclear magnetic resonance confirmed the chemical structure of the product. The associated R9 polypeptide (RRRRRRRRR) was synthesized in large quantities using a polypeptide synthesizer (APEX396, AAPP TEC, Louisville, KY, USA) following standard fluoroen-9-ylmethoxycarboxyl (F-moc) chemistry.

Example 2 stability assay of R9-SSPEI-lncgAS nanoparticle complexes

The stability of the nanocomplexes was determined by qRT-PCR experiment (figure 2). The stability of lncGAS was observed by incubating 50nM R9-SSPEI/lncGAS nanoparticle complexes and 50nM lncGAS (control) with serum at 37 ℃ and performing qRT-PCR on samples at different time points.

Example 3 cytotoxicity assay of R9-SSPEI-lncgAS nanoparticle complexes

MTT experiments were performed after adding R9-SSPEI/lncgAS complexes with different mass ratios (R9-SSPEI/lncgAS mass ratios of 0.5, 1, 2, 4, 8, 16) to the Hela and Siha cells of the cervical cancer cell lines and incubating for 48 hours, and cell proliferation was detected, as shown in FIG. 3.

Example 4 cell culture

The cervical cancer cell lines Hela and Siha were purchased from ATCC cell bank. Establishment of chemotherapy-resistant cervical cancer cell lines Hela/R and Siha/R: hela and Siha cells are irradiated with 2Gy radiation, most of the cells are apoptotic, a small part of the surviving cells are subcultured for 3-5 days, and then the radiation irradiation is performed again, and the steps are repeated circularly until the cells have the survival rate of 60% -90% after receiving the radiation irradiation, and the cells are defined as the radiotherapy-resistant cervical cancer cell lines, as shown in FIG. 4.

Example 5 intracellular uptake assay of R9-SSPEI-lncgAS nanoparticle complexes

Marking cervical cancer cells by using CM-Dil dye, and marking the marked cervical cancer cells at 1 x 10⁵The density of (A) was plated in 12-well plates with slides and cultured for 24 hours; simultaneously, the IncGAS and SSPEI marked by FAM are coupled with two carriers of R9-SSPEI to form two polymers, then the two polymers are respectively added into cells to be incubated for 1 hour, then the cells are fixed and stained with a nuclear signal by DAPI, and finally the uptake condition of the IncGAS in the cells is observed by a confocal fluorescence microscope after mounting. To obtain more accurate quantitative results, flow cytometry was used to detect FAM expression levels within cervical cancer cells. Referring to FIG. 5, it can be seen thatCompared with the control group (shown in the left of the figure), R9-SSPEI can be efficiently taken up in cells.

Example 6 Release of R9-SSPEI-lncgAS nanoparticles in cells

Considering that lncGAS must be released from the R9-SSPEI to be functional in the cell, the next step is to examine whether the R9-SSPEI-lncGAS drug delivery system is able to release functional lncGAS in cervical cancer cells. After transfecting tumor cells with FAM-labeled lncGAS nanoparticles, qRT-PCR (primer sequences are shown in Table 1) and a flow cytometer are respectively used for detecting the release of lncGAS in the cells, and the expression conditions of downstream PI3K-AKT and miR-106 are further detected to judge the biological function of lncGAS.

TABLE 1 qRT-PCR reaction primer sequences

Example 7 cervical cancer cell assay

The R9-SSPEI-lnCGAS nano-particles are transfected in radiotherapy sensitive cervical cancer cell lines (Hela and Siha) and radiotherapy resistant cervical cancer cell lines (Hela/R and Siha/R), and then the cells are irradiated by gamma rays with different doses to carry out various cytological detections: detecting the cell proliferation capacity by MTT (methyl thiazolyl tetrazolium) experiment; detecting apoptosis by Annexin V/PI double staining combined with flow cytometry; detecting the cell cycle by flow cytometry; performing immunofluorescence staining detection on gamma-H2 AX foci to confirm intracellular DNA damage repair; WB assay detected protein expression levels of proliferation marker (PCNA or Ki67), apoptosis marker (cleared caspase 3, Bcl2, Bax), and Caveolin-1.

The results are shown in FIG. 6, wherein A is the expression difference B of LncGAS in the cervical cancer cell line (Hela, Siha) and the radiotherapy-tolerant cell line (Hela-R, Siha-R) thereof is the influence on the survival rate of the cell radiotherapy after the lncGAS is over-expressed in the radiotherapy-tolerant cell line Hela-R; c is the influence of the overexpression of lnCGAS in a radiotherapy-tolerant cell line Siha-R on the survival rate of cell radiotherapy; as can be seen from the figure, the expression of the radiotherapy-resistant cell lines (Hela-R, Siha-R) is remarkably reduced compared with the radiotherapy-sensitive cell lines (Hela, Siha) of the cervical cancer. And after MLLT4-AS1 is over-expressed in Hela-R, Siha-R cells, the sensitivity of radiotherapy is obviously enhanced.

Example 8 animal experiments

In order to study the in vivo uptake of the R9-SSPEI-lncGAS nanoparticle complex, two carriers, namely LncGAS and SSPEI, labeled by FAM are coupled with the R9-SSPEI carrier to form two polymers, then the two polymers are injected into a tumor-bearing mouse body in an intravenous injection mode (the other group is injected with physiological saline as a control), after four hours, the animal is killed, tumor tissues and main metabolic organs (liver, spleen, kidney, heart, brain, lung and ovary) are taken, and the distribution of the two nanoparticle complexes in the organs is observed through a fluorescence imaging system; after the intake experiment is finished, homogenizing the tumor tissues and the main metabolic organ tissues of each group, detecting the release of lncRNA in vivo, and simultaneously reserving part of the tumor tissues and the main metabolic organ tissues to detect the apoptosis condition of the cells in each group by a TUNEL method and an immunohistochemical method.

The results are shown in fig. 7, different vectors carrying biotin labeled lncGAS rat tail artery injection cervical cancer in situ model mice, 4h later sacrifice mice and immediately remove important organs. The results of the small animal imaging system showed that R9-SSPEI/lncGAS was specifically taken up in tumor tissues compared to the control group.

Further for the modulating effect of the R9-SSPEI-lncgAS nanoparticle complex on the in vivo tumor radiotherapeutic sensitivity, a radiotherapeutic sensitive and radiotherapeutic resistant cervical cancer cell line (2X 10) was first used⁶) Constructing a tumor-bearing mouse, injecting a nano-composite of R9-SSPEI-lncGAS and R9-SSPEI-CTR (lncGAS out of order) into the animal subcutaneously after the diameter of a tumor body reaches 8mm (taking another group of injected physiological saline as a contrast), simultaneously carrying out radiation irradiation with the dose of 16Gy, and recording the volume size of the tumor and the survival condition of the animal every week; tumors were harvested after animal death, weighed, and then examined histologically for lncGAS expression and apoptosis-related factor protein expression levels.

The results are shown in FIG. 8, R9-SSPEI/lncGAS nanoparticles can inhibit the growth of tumor in the abdominal cavity, and lncGAS is highly expressed in the R11-SSPEI-miR-145 nanoparticle treated group. The experimental results are expressed as mean ± standard deviation (mean ± SD), statistical analysis is performed by SPSS 19.0 software, single-factor analysis of variance and t-test are adopted for mean comparison among groups, and the P <0.05 on both sides is the significance test standard.

The results of the invention are combined to show that R9-SSPEI-lncGAS provides a novel efficient polypeptide targeting nano-drug delivery system for treating cervical cancer, and has important significance on the treatment strategies of cervical cancer radiotherapy tolerance and tumor recurrence. The targeted medicine for enhancing the tumor radiotherapy sensitivity disclosed by the invention can enter cervical cancer specifically and efficiently, and improves the radiotherapy sensitivity and reduces the radiotherapy side effect by regulating the PI3K/AKT way and exerting the action of microRNA (ribonucleic acid) molecular sponge. The cationic polymer-Polyethyleneimine (PEI) is one of the most widely studied non-viral nano-carriers in recent years, a non-covalent polyelectrolyte complex can be formed by compressing DNA and RNA, and the small nano-particle size and surface positive charge of the complex are favorable for endocytosis of the complex. PEI is considered as an ideal drug delivery carrier because of the characteristics of higher transfection efficiency, low immune response, good biocompatibility and the like. However, the major bottlenecks affecting the application of PEI are its high cytotoxicity and lack of targeting. To address this difficulty, the present invention performs the work of modifying and modifying PEI: modifying PEI by using a small molecular disulfide bond (-SS-), so that the toxicity of PEI is greatly reduced; meanwhile, the colloidal stability of the PEI-nucleic acid complex is improved, and the circulation time of the PEI-nucleic acid complex in vivo is prolonged; meanwhile, the PEI-nucleic acid complex is modified by the tumor targeting polypeptide R9, so that the targeting problem of the nanoparticles in gene therapy research is solved. Therefore, REG is expected to be developed as an effective means for enhancing the sensitivity of radiotherapy for cervical cancer.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A targeted cell-penetrating peptide for enhancing the sensitivity of cervical cancer radiotherapy is characterized in that the targeted cell-penetrating peptide is R9 polypeptide, and the amino acid sequence of the targeted cell-penetrating peptide is shown in SED.ID.NO. 1.

2. A targeted cell-penetrating peptide nano drug delivery system for enhancing the sensitivity of cervical cancer radiotherapy is characterized in that the nano drug delivery system is formed by coupling R9 cell-penetrating peptide of claim 1 to polyethyleneimine PEI through disulfide bond SS.

3. A targeted cell-penetrating peptide nanoparticle complex for enhancing cervical cancer radiotherapy sensitivity is characterized in that the nanoparticle complex is constructed by the drug delivery nano-system R9-SS-PEI of claim 2 carrying lncRNA GAS 5; wherein: lncRNAGAS5 is lncRNA encoded by prostate cancer-associated locus lq 25.

4. Use of the targeted cell-penetrating peptide nano drug delivery system of claim 2 or the targeted cell-penetrating peptide nano particle complex of claim 3 in the preparation of a medicament for treating cervical cancer.

5. The use of claim 4, wherein the medicament is a medicament having a targeting effect for cervical cancer.

6. The use of claim 4, wherein the medicament is a medicament for increasing sensitivity to radiation therapy for cervical cancer.

7. The use of claim 4, wherein the agent is an agent that positively modulates the expression level of PI3K/Akt pathway, PTEN pathway, or miR-106.

8. The use of the targeted cell-penetrating peptide nano drug delivery system of claim 2 or the targeted cell-penetrating peptide nano particle complex of claim 3 in the preparation of a cervical cancer radiation sensitizer.