阅读说明：本技术 一种gsh响应型吉西他滨纳米粒子及其制备方法与应用 (GSH response type gemcitabine nano-particle and preparation method and application thereof ) 是由 林东军 张鑫宇 钟文浩 于 2021-07-27 设计创作，主要内容包括：本发明公开了一种GSH响应型吉西他滨聚合物,由亲水性聚合物、含两个羧基的二硫键化合物以及吉西他滨反应制备而成。其中,含两个羧基的二硫键化合物与吉西他滨一步反应合成含二硫键的吉西他滨前药,封闭固定吉西他滨药物结构的活性基团,提高药物的稳定性,减小药物与正常细胞DNA结合机率。本发明还提供了一种由GSH响应型吉西他滨聚合物、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000组成的GSH响应型吉西他滨纳米粒子。所述GSH响应型吉西他滨纳米粒子的血液相容性良好,能够抑制B细胞淋巴瘤细胞增殖,且具有氧化还原响应性,能够在高浓度GSH肿瘤微环境的刺激下可控释放吉西他滨,提高药物对肿瘤的疗效,减小药物对机体毒副反应。(The invention discloses a GSH response type gemcitabine polymer, which is prepared by the reaction of a hydrophilic polymer, a disulfide bond compound containing two carboxyl groups and gemcitabine. Wherein, the disulfide bond compound containing two carboxyl groups and gemcitabine react in one step to synthesize the gemcitabine prodrug containing the disulfide bond, and the active group for fixing the gemcitabine drug structure is sealed, so that the stability of the drug is improved, and the combination probability of the drug and normal cell DNA is reduced. The invention also provides GSH response type gemcitabine nano particles consisting of the GSH response type gemcitabine polymer and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000. The GSH response type gemcitabine nano particles have good blood compatibility, can inhibit the proliferation of B cell lymphoma cells, has redox responsiveness, can controllably release gemcitabine under the stimulation of a high-concentration GSH tumor microenvironment, improves the curative effect of a drug on tumors, and reduces the toxic and side effects of the drug on organisms.)

1. A GSH response type gemcitabine polymer is characterized in that the GSH response type gemcitabine polymer is prepared by the reaction of a hydrophilic polymer, a disulfide bond compound containing two carboxyl groups and gemcitabine; the structural formula of the GSH-responsive gemcitabine polymer is shown in the formula (I):

wherein R2 is selected from CH₂、CH₂-CH₂Or CH₂-CH₂-CH₂(ii) a R1, R3 or R4 is a hydrophilic polymer or gemcitabine or hydroxyl, and at least one of R1, R3 or R4 is a hydrophilic polymer; the hydrophilic polymer is PEG or mPEG-OH, mPEG-NH₂。

2. The GSH-responsive gemcitabine polymer of claim 1,

r1, R3 or R4 are or-OH, and at least one of R1, R3 or R4 is

3. The GSH-responsive gemcitabine polymer of claim 1, having a number average molecular weight of 3000 to 10000; the number average molecular weight of the hydrophilic polymer is 500-2000.

4. The GSH-responsive gemcitabine polymer of claim 1, wherein the molar ratio of said hydrophilic polymer, said disulfide compound having two carboxyl groups, and gemcitabine is (0.1-0.5): (1-4.5): 1.

5. a method of preparing a GSH-responsive gemcitabine polymer of any one of claims 1 to 4 comprising the steps of:

s1, mixing gemcitabine, a disulfide bond compound HOOC-R2-S-S-R2-HOOC containing two carboxyl groups, DCC and DMAP, dissolving in an organic solvent, and reacting for 18-36 hours to obtain a disulfide bond-containing gemcitabine prodrug solution; r2 is selected from CH₂、CH₂-CH₂Or CH₂-CH₂-CH₂；

S2, adding the hydrophilic polymer, DCC and HOBT into the disulfide bond-containing gemcitabine prodrug solution in the step S1, reacting for 24-48 h, filtering the product, taking the filtrate, dialyzing, and performing rotary evaporation, glacial ethyl ether precipitation, suction filtration and vacuum drying to obtain GSH response type gemcitabine polymer powder.

6. The method according to claim 5, wherein the molar ratio of the disulfide compound having two carboxyl groups, DCC and DMAP in step S1 is 1: (2-2.4): (0.1-0.6).

7. The method of claim 6, wherein the molar ratio of the hydrophilic polymer, HOBT and DCC in step S2 is 1: (0.74-1.1): (0.45-0.61).

8. A GSH-responsive gemcitabine nanoparticle comprising the GSH-responsive gemcitabine polymer as claimed in any one of claims 1 to 4, and distearoylphosphatidylethanolamine-polyethylene glycol 2000, wherein the mass ratio of the GSH-responsive gemcitabine polymer to the distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 1: (0.1-0.8).

9. The method of preparing GSH-responsive gemcitabine nanoparticles of claim 8, comprising the steps of: dissolving the GSH-responsive gemcitabine polymer of any one of claims 1 to 4 in a solvent, adding distearoylphosphatidylethanolamine-polyethylene glycol 2000 after fully dissolving, dripping into water after mixing, stirring, filtering, centrifuging, and removing impurities to obtain the GSH-responsive gemcitabine polymer.

10. Use of the GSH-responsive gemcitabine nanoparticles prepared by the method of claim 9 in the preparation of an anti-tumor drug.

Technical Field

The invention relates to the technical field of biological medicines, in particular to GSH (glutathione) response type gemcitabine (Gemcitabine) nanoparticles and a preparation method and application thereof.

Background

Gemcitabine is a hydrophilic cytosine nucleoside derivative, which is activated by deoxycytidine kinase after entering human body, and metabolized by cytosine nucleoside deaminase, and its main metabolite is incorporated into DNA in cells, and mainly acts on tumor cells during DNA synthesis. Gemcitabine, therefore, is a difluoronucleoside antimetabolite anticancer agent that disrupts tumor cell replication.

In recent years, based on the unique properties of tumor microenvironment (such as high concentration Glutathione (GSH), low pH), a series of functionalized polymer drug conjugates have been developed for tumor microenvironment response drug administration, and great progress has been made in drug targeted delivery, controlled release, improved therapeutic effect, and the like. The prepared tumor microenvironment responsive nano drug-loaded system is very sensitive to the stimulation of tumor tissues, can selectively release drugs at tumor positions, not only can improve the curative effect of the drugs, but also can effectively reduce the toxic and side effects of the drugs on normal cells of organisms. In the research of the tumor microenvironment response type nano drug delivery system, the construction of the redox response drug delivery system is one of the hot points of research. The existing research results show that the concentration (0.5-10 mmol/L) of GSH in tumor cells is significantly higher than that (2-20 mu mol/L) of GSH in normal cells, and disulfide bonds are rapidly cracked under the action of the high-concentration GSH, so that the disulfide bonds are widely used as an ideal trigger of a redox response drug delivery system. Currently, there are many studies on the loading of a redox-responsive drug delivery system with gemcitabine drug for tumor therapy, such as: chen et al constructed a reductively-responsive crosslinked gemcitabine prodrug micelle, which is a copolymer copolymerized of poly (ethylene glycol) methacrylate (PEGMA), 7- (2-methacryloylethoxy) -4-methylcoumarin (CMA) and 2- ((2-hydroxyethyl) disulfanyl) acrylate ethyl ester-gemcitabine (HSEA-GEM), having "stealth" surface, photocrosslinking properties and reduction sensitivity, and is effective in inhibiting the proliferation of BxPC-3 pancreatic cancer cells (Chen X, et al, one-step prediction of reduction-responsive cross-linking-pancreatic cancer cells for intracellular drug delivery [ J ]. Colloids and Surfaces B: Biointerfaces,2019,181: 94-101); sun et al constructed a gemcitabine-based small vector co-delivered triple drug, specifically: experimental results of using redox-responsive Gemcitabine (GEM) -conjugated polymer PGEM as tumor penetrating nanocarrier to Co-carry immunomodulators (NLG919, inhibitors of indoleamine 2, 3-dioxygenase 1(IDO 1)) and chemotherapeutic Drugs (paclitaxel (PTX) for combined immunotherapy and chemotherapy) show that the Small Carrier Co-delivers triple drug to penetrate deep into the core of Pancreatic tumor and show better antitumor activity (Sun J, et al. triple Drugs Co-deleted by a. Small-molecule chemotherapeutics base-Carrier for Pancreatic Cancer Imnochemithermology [ J ]. Acta biomaterials, 2020,106:289-300) DNA binding remains a difficult problem to solve.

Disclosure of Invention

The present invention is directed to overcoming the above-mentioned drawbacks and deficiencies of the prior art and providing a GSH-responsive gemcitabine polymer.

The second objective of the present invention is to provide a method for preparing the GSH-responsive gemcitabine polymer.

The third objective of the present invention is to provide a GSH-responsive gemcitabine nanoparticle.

The fourth object of the present invention is to provide a method for preparing the GSH-responsive gemcitabine nanoparticles.

The fifth purpose of the present invention is to provide an application of the GSH-responsive gemcitabine nanoparticles prepared by the above preparation method in preparing an antitumor drug.

The above object of the present invention is achieved by the following technical solutions:

a GSH response type gemcitabine polymer is prepared by the reaction of a hydrophilic polymer, a disulfide bond compound containing two carboxyl groups and gemcitabine; the structural formula of the GSH-responsive gemcitabine polymer is shown in the formula (I):

formula (I);

wherein R2 is selected from CH₂、CH₂-CH₂Or CH₂-CH₂-CH₂(ii) a R1, R3 or R4 is a hydrophilic polymer or gemcitabine or hydroxyl, and at least one of R1, R3 or R4 is a hydrophilic polymer; the hydrophilic polymer is PEG or mPEG-OH, mPEG-NH₂。

Preferably, R1, R3 or R4 is or-OH, and at least one of R1, R3 or R4 is

In GSH response type gemcitabine polymer (GEM-S-S-PEG), disulfide compound containing two carboxyl groups reacts with amino and/or hydroxyl of gemcitabine to block and fix gemcitabine molecule, thus enhancing stability of gemcitabine in vivo and reducing probability of binding with DNA of normal tissues in vivo. In addition, the disulfide compound containing two carboxyl groups and the hydrophilic polymer are subjected to esterification or condensation reaction, so that gemcitabine molecules are further blocked and fixed, and the biocompatibility of gemcitabine is improved. The GEM-S-S-PEG provided by the invention effectively inhibits the release and accumulation of gemcitabine in normal tissue parts through the double blocking and fixing effects, so that the toxic and side effects of the gemcitabine on normal tissues are reduced.

Preferably, the disulfide compound having two carboxyl groups includes any one of 2,2' -thiodiacetic acid, 3' -thiodipropionic acid, and 4,4' -thiodibutanoic acid.

Preferably, the number average molecular weight of the GEM-S-S-PEG is 3000-10000.

Preferably, the number average molecular weight of the GEM-S-S-PEG is 3500-6000.

Preferably, the number average molecular weight of the hydrophilic polymer is 500 to 2000.

More preferably, the hydrophilic polymer has a number average molecular weight of 2000.

Preferably, the molar ratio of the hydrophilic polymer, the disulfide compound containing two carboxyl groups and gemcitabine is (0.1-0.5): (1-4.5): 1.

the molar weight of the hydrophilic polymer of the present invention is the ratio of the mass of the hydrophilic polymer to the number average molecular weight.

The invention also provides a preparation method of any one of the GSH-responsive gemcitabine polymers, which comprises the following steps:

s1, mixing gemcitabine, a disulfide bond compound HOOC-R2-S-S-R2-HOOC containing two carboxyl groups, DCC and DMAP, dissolving in an organic solvent, and reacting for 18-36 hours to obtain a disulfide bond-containing gemcitabine prodrug solution; r2 is selected from CH₂、CH₂-CH₂Or CH₂-CH₂-CH₂；

S2, adding the hydrophilic polymer, DCC and HOBT into the gemcitabine prodrug solution containing the disulfide bond in the step S1, reacting for 24-48 h, filtering a product, taking a filtrate, dialyzing, and performing rotary evaporation, glacial ethyl ether precipitation, suction filtration and vacuum drying to obtain GEM-S-S-PEG powder.

Preferably, the molar ratio of the disulfide compound having two carboxyl groups, DCC, DMAP in step S1 is 1: (2-2.4): (0.1-0.6).

Preferably, the mole ratio of the hydrophilic polymer, HOBT and DCC in step S2 is 1: (0.74-1.1): (0.45-0.61).

Preferably, the step of dialysis in step S2 is: first dialyzed against DMF for 3 days and then against water for 3 days.

The invention also provides GSH-responsive gemcitabine nanoparticles (GSP NPs), which consist of any one of the GSH-responsive gemcitabine polymers and distearoylphosphatidylethanolamine-polyethylene glycol 2000, wherein the mass ratio of the GSH-responsive gemcitabine polymer to the distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 1: (0.1-0.8).

The invention also provides a preparation method of the GSH-responsive gemcitabine nanoparticles, which comprises the following steps: dissolving the GSH response type gemcitabine polymer in a solvent, adding distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000(DSPE-PEG2000) after fully dissolving, dripping into water after uniformly mixing, stirring, filtering, centrifuging, and removing impurities to obtain the GSH response type gemcitabine polymer.

Preferably, the solvent is DMSO.

As a preferred embodiment, a method for preparing GSH-responsive gemcitabine nanoparticles comprises the following steps:

s1, adding 2.84mmol of gemcitabine and 4.04mmol of 3,3' -dithiodipropionic acid into a mixed solution of 9.11mmol of DCC and 1.95mmol of DMAP, and reacting for 18h to obtain a disulfide bond-containing gemcitabine prodrug solution;

s2, adding 0.4mmol of mPEG-OH with the number average molecular weight of 2000, 0.37mmol of HOBT and 0.24mmol of DCC into the gemcitabine prodrug solution containing the disulfide bond synthesized in the step S1, reacting for 24 hours, filtering a product, taking a filtrate, dialyzing in DMF for 3 days (the DMF is replaced for three times), continuously dialyzing in water for 3 days, and performing rotary evaporation, glacial ethyl ether precipitation, suction filtration and vacuum drying to obtain GEM-S-S-PEG powder;

s3, sufficiently dissolving the GEM-S-S-PEG solid powder obtained in the step S2 in DMSO to prepare a GEM-S-S-PEG solution with the concentration of 20mg/mL, uniformly mixing 300 mu L of GEM-S-S-PEG solution, 60 mu L of DSPE-PEG2000 solution with the concentration of 20mg/mL and 240 mu L of DMSO solution, dropwise adding into 18mL of ultrapure water, and stirring for 1min by using a magnetic stirrer. Filtering, centrifuging at 2500rpm for 15min, repeatedly centrifuging for 3 times, and removing impurities to obtain GSP NPs.

The invention also provides application of the GSH response type gemcitabine nano particles prepared by the preparation method in preparing antitumor drugs.

Preferably, the tumor is a B cell lymphoma.

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

(1) the GSH response type gemcitabine polymer provided by the invention is prepared from a hydrophilic polymer, a disulfide bond compound containing two carboxyl groups and gemcitabine, wherein the disulfide bond compound containing two carboxyl groups is used as a bridge for connecting gemcitabine molecules, and the two disulfide bond compounds react to obtain a disulfide bond-containing gemcitabine prodrug, so that an active group for sealing and fixing a gemcitabine drug structure is closed, the stability of the gemcitabine drug is improved, and the probability of combining the gemcitabine drug with normal cell DNA is reduced; in addition, the disulfide compound containing two carboxyl groups reacts with gemcitabine molecules, and the remaining carboxyl groups further undergo esterification or condensation reaction with hydrophilic polymers, so that gemcitabine molecules are further blocked and fixed, and the biocompatibility of gemcitabine is improved.

(2) The invention also provides GSH response type gemcitabine nano-particles consisting of the GSH response type gemcitabine polymer and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, the nano-particles have good blood compatibility, can inhibit the proliferation of lymphoma cells, have redox responsiveness, and can controllably release drugs under the stimulation of a high-concentration GSH tumor microenvironment, reduce the toxic and side reactions of the drugs to organisms and improve the curative effect of the drugs to tumors.

Drawings

FIG. 1 shows the NMR of GEM-S-S-PEGSpectrum¹H-NMR chart.

FIG. 2 is the NMR spectrum of gemcitabine¹H NMR chart.

FIG. 3 is a mass spectrum of GEM-S-S-PEG.

FIG. 4 is a particle size distribution diagram of GSP NPs.

FIG. 5 is a Zeta potential diagram of GSP NPs.

FIG. 6 is a TEM image of GSP NPs.

Fig. 7 shows the particle size variation of GSP NPs in different solvents.

FIG. 8 shows the change in PDI of GSP NPs in different solvents.

FIG. 9 shows the hemolysis ratio of GSP NPs (C +: positive, C1: 250. mu.g/mL, C2: 125. mu.g/mL, C3: 62.5. mu.g/mL, C4: 31.25. mu.g/mL, C5: 15.625. mu.g/mL, C6: 7.8125. mu.g/mL).

FIG. 10 is a CCK8 method for determining the in vitro cell activity of gemcitabine and GSP NPs on A20 cells.

Figure 11 is the result of the induction of a20 apoptosis by free gemcitabine and GSP NPs.

FIG. 12 is a graph showing the biodistribution of NPs @ DiR and free DiR in A20 tumor-bearing mice.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

EXAMPLE 1 preparation of GSP NPs

S1, adding 2.84mol of gemcitabine and 4.04mol of 3,3' -dithiodipropionic acid into a mixed solution of 9.11mmol of DCC and 1.95mmol of DMAP, and reacting for 18h to obtain a disulfide bond-containing gemcitabine prodrug solution.

S2, adding 0.4mmol of mPEG-OH with the number average molecular weight of 2000, 0.37mmol of HOBT and 0.24mmol of DCC into the gemcitabine prodrug solution containing the disulfide bond synthesized in the step S1, reacting for 24 hours, filtering a product, taking a filtrate, dialyzing in DMF for 3 days (the DMF is replaced for three times), continuously dialyzing in water for 3 days, and performing rotary evaporation, glacial ethyl ether precipitation, suction filtration and vacuum drying to obtain GEM-S-S-PEG.

The structural formula of the GEM-S-S-PEG is as follows:

s3, sufficiently dissolving the GEM-S-S-PEG solid powder obtained in the step S2 in DMSO to prepare a GEM-S-S-PEG solution with the concentration of 20mg/mL, uniformly mixing 300 mu L of GEM-S-S-PEG solution, 60 mu L of DSPE-PEG2000 solution with the concentration of 20mg/mL and 240 mu L of DMSO solution, dropwise adding into 18mL of ultrapure water, and stirring for 1min by using a magnetic stirrer. Filtering, centrifuging at 2500rpm for 15min, repeatedly centrifuging for 3 times, and removing impurities to obtain GSP NPs.

EXAMPLE 2 preparation of GSP NPs

S1, adding 1mmol of gemcitabine and 2mmol of 3,3' -dithiodipropionic acid into a mixed solution of 4mmol of DCC and 0.2mmol of DMAP, and reacting for 18h to obtain a disulfide bond-containing gemcitabine prodrug solution.

S2, adding 0.2mmol of mPEG-OH with molecular weight (number average) of 2000, 0.15mmol of HOBT and 0.1mmol of DCC into the gemcitabine prodrug solution containing disulfide bonds synthesized in the step S1, reacting for 24h, filtering a product, taking a filtrate, dialyzing in DMF for 3 days (changing DMF for three times), continuously dialyzing in water for 3 days, and performing rotary evaporation, glacial ethyl ether precipitation, suction filtration and vacuum drying to obtain GEM-S-S-PEG.

S3, sufficiently dissolving the GEM-S-S-PEG solid powder obtained in the step S2 in DMSO to prepare a GEM-S-S-PEG solution with the concentration of 20mg/mL, uniformly mixing 200 mu L of GEM-S-S-PEG solution, 160 mu L of DSPE-PEG2000 solution with the concentration of 20mg/mL and 240 mu L of DMSO solution, dropwise adding into 18mL of ultrapure water, and stirring for 2min by using a magnetic stirrer. Filtering, centrifuging at 2500rpm for 15min, repeatedly centrifuging for 3 times, and removing impurities to obtain GSP NPs.

EXAMPLE 3 preparation of GSP NPs

S1, adding 2mmol of gemcitabine and 3mmol of 3,3' -dithiodipropionic acid into a mixed solution of 7.2mmol of DCC and 1.8mmol of DMAP, and reacting for 36h to obtain a disulfide bond-containing gemcitabine prodrug solution.

S2, adding 0.3mmol of mPEG-OH with molecular weight (number average) of 2000, 0.3mmol of HOBT and 0.14mmol of DCC into the gemcitabine prodrug solution containing disulfide bonds synthesized in the step S1, reacting for 48 hours, filtering a product, taking a filtrate, dialyzing in DMF for 3 days (changing DMF for three times), continuously dialyzing in water for 3 days, and performing rotary evaporation, glacial ethyl ether precipitation, suction filtration and vacuum drying to obtain GEM-S-S-PEG.

S3, sufficiently dissolving the GEM-S-S-PEG solid powder obtained in the step S2 in DMSO to prepare a GEM-S-S-PEG solution with the concentration of 20mg/mL, uniformly mixing 100 mu L of GEM-S-S-PEG solution, 60 mu L of DSPE-PEG2000 solution with the concentration of 20mg/mL and 120 mu L of DMSO solution, dropwise adding 9mL of ultrapure water, and stirring for 3min by using a magnetic stirrer. Filtering, centrifuging at 2500rpm for 15min, repeatedly centrifuging for 3 times, and removing impurities to obtain GSP NPs.

Example 4 preparation of GSP NPs

GSP NPs were prepared by following the same procedure as in example 1 except that "3, 3 '-dithiodipropionic acid" in step S1 of example 1 was replaced with "2, 2' -thiodiacetic acid".

EXAMPLE 5 preparation of GSP NPs

GSP NPs were prepared by following the same procedure as in example 1 except that "3, 3 '-dithiodipropionic acid" in step S1 of example 1 was replaced with "4, 4' -thiodibutanoic acid".

EXAMPLE 6 preparation of GSP NPs

Only "mPEG-OH" at step S2 in example 1 was replaced with "mPEG-NH₂", the remaining steps were the same as in example 1, to prepare GSP NPs.

Example 7 preparation of GSP NPs

Only the "3, 3' -dithiodipropionic acid" in step S1 and the "mPEG-OH" in step S2 were replaced with "mPEG-NH" in example 1₂", the remaining steps were the same as in example 1, to prepare GSP NPs.

Test example 1 characterization of GSP NPs

1. Nuclear magnetic resonance hydrogen spectrum (¹H-NMR) analysis

The disulfide bond-containing gemcitabine prepared in step S1 of example 1 and GEM-S-S-PEG prepared in step S2 were characterized by hydrogen nuclear magnetic resonance spectroscopy.

And (4) analyzing results: of the final product GEM-S-S-PEG¹H NMR chart Gemcitabine¹The H NMR chart is shown in FIG. 2. As can be seen from FIGS. 1 and 2, of GEM-S-S-PEG¹Peaks at d and f in the H NMR spectrum are shifted, the peak position at d is changed from delta 4.20-4.26 ppm (figure 2) to delta 5.42-5.58 ppm (figure 1), and the peak position at f is changed from delta 3.60-3.70 ppm (figure 2) to delta 4.30-4.50 ppm (figure 1). The o-OH peaks at the f and d sites completely disappeared, demonstrating that there was no free gemcitabine in the final product. To further confirm the newly formed structure, the results showed that in GEM-S-S-PEG¹H NMR spectrum (FIG. 1) of-NH on gemcitabine structure at delta 7.40-7.48 ppm (FIG. 2)₂The peak disappeared and a new-NHCO-peak (delta 8.20-8.30 ppm) appeared, indicating that three different sites of gemcitabine were reacted. Also shown in FIG. 1 is-CH₂an-S-peak (. delta.0.80-1.82 ppm), no-COOH peak, confirming successful grafting of 3,3 '-dithiodipropionic acid to the product, and no residual free 3,3' -dithiodipropionic acid.

2. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

The GEM-S-PEG prepared in step S2 of example 1 was detected using a matrix assisted laser desorption/ionization time-of-flight mass spectrometer.

And (4) analyzing results: the mass spectrum of GEM-S-S-PEG is shown in FIG. 3, and it can be seen from FIG. 3 that the molecular weight distribution of GEM-S-S-PEG is 3500-4600, and the gemcitabine loading ratio in GEM-S-S-PEG is 24.6% according to the intensity of proton absorption peak.

Test example 2 particle size distribution, potential and stability of GSP NPs

Particle size distribution and potential of GSP NPs

The particle size distribution and Zeta potential of the GSP NPs prepared in example 1 were measured using a dynamic light scattering particle sizer (DLS) and characterized using a Transmission Electron Microscope (TEM).

And (4) analyzing results: the particle size distribution and potential of GSH-responsive gemcitabine nanoparticles (GSP NPS) are shown in FIGS. 4 and 5, respectively, and the average particle size of GSP NPs is 83.25nm, and the potential is about-19.4 mV. FIG. 6 is a TEM image of GSP NPs, from which it can be seen that a fraction of the nanoparticles have a slightly smaller size (about 50nm) as measured by TEM than the GSP NPs as measured by DLS, probably because the mPEG hydrophilic shell of the nanoparticles shrinks, resulting in a smaller particle size of the nanoparticles, due to the dehydration of the sample during the preparation of the TEM test sample.

Stability of GSP NPs

To explore the stability of GSP NPs, different solvents were selected to dilute the concentrated GSP NPs. The method comprises the following specific steps: GSP NPs at a mass concentration of 500. mu.g/mL were diluted to 50. mu.g/mL with ultrapure water, 1 XPBS, and 1 XPBS containing 10% fetal bovine serum, respectively, and the particle size distribution and the Polymer Dispersion Index (PDI) thereof were measured by DLS, and the results are shown in FIGS. 7 and 8, respectively. As can be seen from fig. 7, the particle size of the GSP NPs did not change significantly after dilution in ultrapure water, 1 × PBS, and 1 × PBS containing 10% fetal bovine serum for 5 days, indicating that the stability of the GSP NPs was good. As can be seen from FIG. 8, the GSP NPs after 5 days of dilution in different solvents had PDI less than 0.3 and a more uniform particle size distribution.

Test example 3 evaluation of in vitro biological Properties of GSP NPs

1. Hemolysis test

The experimental steps are as follows: fresh blood was taken from healthy SD rats, centrifuged at 3000rpm for 10 minutes, and then red blood cells obtained by centrifugation were further washed with physiological saline until the supernatant was clear. Then, the GSP NPs were diluted with physiological saline to obtain six GSP NPs of different concentrations, 250, 125, 62.5, 31.25, 15.625 and 7.8125 μ g/mL, which were sequentially designated as C1-C6 groups, and mixed with an equal volume of erythrocyte suspension. Meanwhile, physiological saline containing a suspension of erythrocytes was used as a negative control (hemolysis rate of 0%), and an equivalent suspension of erythrocytes diluted with ultrapure water was used as a positive control (hemolysis rate of 100%, and this was designated as C + group). All samples were placed in a constant temperature shaker and incubated at 37 ℃ for 2h with shaking at 100 rpm. After the completion of shaking, the mixture was centrifuged at 3000rpm for 10 minutes, photographed, and the supernatant was collected, and the absorbance of each group was measured at 436nm with a microplate reader to analyze the hemolysis rate. Experiments were performed in triplicate.

And (4) analyzing results: the results of the hemolysis rate test of GSP NPs are shown in FIG. 9. As can be seen from the figure, the color of the red blood cell suspension of the GSP NPs-treated group did not show obvious hemolysis with the increase of the drug concentration compared with the positive control group. The hemolysis rate of GSP NPs is still less than 5% when the concentration is as high as 250. mu.g/mL. The above results indicate that GSP NPs have good blood compatibility.

2. Mouse B cell lymphoma cell A20 cytotoxicity

The cytotoxicity of free gemcitabine and GSP NPs was tested using CCK8 kit. A20 cell (1.5X 10)⁴Cells/well) were inoculated in 96-well plates, and different concentrations (0.005, 0.02, 0.1, 2, 10, 50. mu.g/mL) of free gemcitabine and GSP NPs were added, placed at 37 ℃ with 5% CO₂Culturing in an incubator for 24 h. After incubation, 10. mu.L of CCK8 solution was added to each well. After 4 hours, the absorbance value of each well was analyzed at 450nm using a microplate reader. The blank culture medium is a zero setting group, the cell group without drug treatment is a control group, and three parallel groups are set in each group of experiments.

And (4) analyzing results: the results of the CCK8 method for determining the in vitro cell activities of gemcitabine and GSP NPs on a20 cells are shown in fig. 10. The free gemcitabine and the GSP NPs can inhibit cell proliferation, and the proliferation inhibition of the GSP NPs on A20 cells is gradually enhanced along with the increase of the concentration of the drugs, which shows that the in vitro antiproliferative activity of the two drugs has concentration and time dependence. The experimental result shows that when the concentration of the medicine reaches 50 mu g/mL, the GSP NPs have better inhibition effect on the activity of A20 cells compared with the free gemcitabine.

Apoptosis of A20 cells

The apoptosis induced by GSP NPs was detected using annexin V-FITC/PI apoptosis kit. A20 cell (3.0X 10)⁴Cells/well) were seeded in 6-well plates and then treated with free gemcitabine, GSP NPs (gemcitabine equivalent concentration 2.5 μ g/mL), respectively, and the non-dosed cell group was the control group. After 24h, the medium was discarded, the cells were washed with PBS, and the cells were collected; then suspending 500 mu L Binding Buffer cells, adding 5 mu L Annexin V-FITC and 10 mu L Propidium Iodide, mixing uniformly, and incubating for 5-15 min at room temperature in a dark place; and finally, carrying out flow cytometry detection.

And (4) analyzing results: the results of the induction of apoptosis of a20 cells by free gemcitabine and GSP NPs are shown in fig. 11. As shown, the total apoptosis rate of GSP NPs group a20 cells was slightly higher than that of free gemcitabine group a20 cells after 48h of action. It is shown that GSP NPs can effectively induce apoptosis of A20 cells.

Test example 4 in vivo biodistribution of GSP NPs

The biodistribution of GSP NPs in vivo was determined by a small animal in vivo imaging system. The biodistribution of GSP NPs was studied in A20 tumor-bearing mice. Female BALB/c Nude mice (8 weeks, 20g) were injected with 100. mu.L of A20 cell suspension subcutaneously in hind limbs (cell number 5.0X 10)⁶). When the tumor volume is increased to 300-400 mm³Thereafter, A20 tumor-bearing mice were randomized into two groups, and free DiR and fluorescein-DIR-entrapped GSP NPs (NPs @ DiR, DiR equivalent concentration 0.5mg/kg) were administered intravenously. All A20 tumor-bearing mice were then systemically anesthetized by intraperitoneal injection of a 1% sodium pentobarbital solution (50 mg/kg). After successful anesthesia, a20 tumor-bearing mice were placed in a small animal in vivo imaging system and the fluorescence distribution was observed at 1, 4, 8, 12, 24, 48, and 72 hours after intravenous injection of free DiR, NPs @ DiR, respectively.

And (4) analyzing results: the biodistribution of NPs @ DiR and free DiR in A20 tumor-bearing mice is shown in FIG. 12. After the tumor-bearing mice are injected with the free DiR, the fluorescence imaging signals of the tumor parts are weak, while the tumor parts of the tumor-bearing mice injected with the NPs @ DiR show stronger fluorescence intensity, the fluorescence enrichment of the tumor parts can be observed in 1h after the injection, the fluorescence intensity of the tumor parts is gradually enhanced along with the time extension, the fluorescence intensity is strongest in 12h, and the enrichment level of the NPs @ DiR at the tumor parts is highest at the moment. The above results indicate that NPs @ DiR has a stronger enrichment capacity at the tumor site than free DiR.