Acid-activated transmembrane polypeptide carrier and use thereof

Abstract

The application discloses an acid-activated transmembrane polypeptide carrier and belongs to the technical field of biological medicines. a H b X n The arrangement order of the three kinds of amino acids is not limited; wherein X is a positive charge amino acid and an analogue; a=5, 6, 7, 8, 9, 10; b=4, 5, 6, 7, 8, 9; n=0, 1, 2, 3, 4. The polypeptide carrier has a simple structure, and cell uptake experiments under different pH conditions, in-vitro anti-tumor experiments and hemolysis experiments all show that the designed and synthesized polypeptide carrier L a H b X n has obvious acid-activated transmembrane activity, low toxicity, can effectively carry an anti-tumor drug CPT into a tumor cell to exert anti-tumor activity and shows tumor targeting. In addition, the polypeptide shows rapid membrane breaking activity after carrying the CPT, so the polypeptide also has great application potential in the preparation of anti-tumor drugs and anti-multiple drug resistance. Therefore, the polypeptide has good application prospect in the preparation of clinical application drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a class of targeted polypeptide carriers with acid-activated cell membrane penetration activity. This invention also includes the application of the acid-activated membrane-penetrating polypeptide carrier in the preparation of drug delivery systems, anti-tumor drug delivery, and anti-multidrug resistance. Background Technology

[0002] In recent years, transmembrane peptides (CPPs) have attracted widespread attention due to their ability to carry various drugs and biomolecules into cells without significant toxicity [Biomaterials., 2013, 7080-7993]. However, most transmembrane peptides are positively charged and interact with the negatively charged cell membrane, resulting in a lack of selectivity, which hinders their in vivo application [Adv. Drug Deliv. Rev., 2005, 529-545; J. Am. Chem. Soc., 2014, 12868-12871].

[0003] To improve the selectivity of transmembrane peptides and reduce their toxic side effects, researchers have proposed many solutions. For example, using cleavable PEG to design nanocarriers can reduce the non-specific permeation of traditional transmembrane peptides. However, PEG chains cannot be effectively and completely cleaved at the tumor site, thus weakening the cellular permeability of CPPs [ACS.Nano., 2012, 6:3491-8; Mol.Pharm., 2010, 7:1816-26]. Furthermore, the abnormal proliferation of tumor cells results in a slightly lower pH value in the environment surrounding the tumor compared to normal tissue [Mol.Pharm., 2011, 8:2032-8], which provides a possibility for designing acid-targeting carriers or drug delivery systems.

[0004] Histidine, a unique amino acid, possesses buffering capacity in biological systems. The pKa value of the imidazole ring in histidine is around 6.5, allowing it to protonate and acquire a positive charge in acidic environments, while remaining uncharged under physiological pH conditions [Protein.Sci.,2006,15:1214-8]. Utilizing this unique property, researchers have designed a series of pH-sensitive, histidine-rich acid-targeting CPPs [Bioconjug.Chem.,2011,22:1410-5].

[0005] TAT-penetrating peptides are a class of short-chain polypeptides rich in basic amino acids discovered in recent years. They have a strong ability to penetrate cell membranes. After being linked with nucleic acid molecules, drug protein molecules, and even viral surface molecules, they can be successfully carried into cells to exert their effects, and are known as "biological missiles". Inspired by Tat-penetrating peptides and oligoarginine peptides, researchers have designed many cationic-penetrating peptides [Chem.Res.,2013,46(12):2944-2954;J.Med.Chem.,2002,45(17):3612-3618;J.Am.Chem.Soc.,2015,137(23):7357-64].

[0006] The above studies all indicate that the number of positive charges has an important influence on the transmembrane activity of transmembrane peptides, and that appropriately increasing the positive charge on the peptide chain can effectively improve its transmembrane activity [AIChE.J,2019,65;J.Drug Target.,2011,19(8):675-680]. Summary of the Invention

[0007] One of the objectives of this invention is to provide a membrane-penetrating polypeptide carrier that is structurally simple, low in toxicity, and can be activated by the acidic environment of a tumor.

[0008] The second objective of this invention is to provide the application of the above-mentioned acid-activated membrane-penetrating polypeptide carrier in the preparation of drug delivery systems.

[0009] The third objective of this invention is to provide the application of the above-mentioned acid-activated membrane-penetrating polypeptide carrier in the preparation of antitumor drugs.

[0010] The fourth objective of this invention is to provide the application of the above-mentioned acid-activated membrane-penetrating polypeptide carrier in the treatment of multidrug resistance.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] (I) Structural design of an acid-activated membrane-penetrating polypeptide carrier

[0013] This invention relates to an acid-activated membrane-penetrating polypeptide carrier, whose structure is rich in leucine, histidine, and an appropriate amount of positively charged amino acids, and whose general structural formula is: L a H b X n (The order of the three amino acids is not specified), among which,

[0014] X = K or other positively charged amino acids and similar substances;

[0015] a = 5, 6, 7, 8, 9, 10;

[0016] b = 4, 5, 6, 7, 8, 9;

[0017] n = 0, 1, 2, 3, 4.

[0018] Specifically, the acid-activated membrane-penetrating polypeptide carrier of the present invention has the following general structural formula: Leu a His b X n (The order of the three amino acids is not specified), among which,

[0019] X = Lys or other positively charged amino acids and analogues;

[0020] a = 5, 6, 7, 8, 9, 10;

[0021] b = 4, 5, 6, 7, 8, 9;

[0022] n = 0, 1, 2, 3, 4.

[0023] As a preferred embodiment of the present invention, the acid-activated membrane-penetrating polypeptide carrier analogs mentioned above include L6H8K0(LH), L6H8K1(LH-K), L6H8K2(LH-2K), L5H9K0(L5H9) and L5H9(K-C6)1(L5H9K-C6), which are denoted as LH, LH-K, LH-2K, L5H9 and L5H9K-C6 respectively, and their amino acid sequences are shown in SEQ ID No. 1 to SEQ ID No. 5 respectively.

[0024] All of the above acid-activated membrane-penetrating polypeptide carriers were prepared using classical solid-phase synthesis methods.

[0025] (II) Study on the transmembrane activity of acid-activated transmembrane peptide carriers

[0026] This invention takes L6H8K0(LH), L6H8K1(LH-K), L6H8K2(LH-2K), L5H9K0(L5H9), and L5H9(K-C6)1(L5H9K-C6) as examples to study the target carrier peptide acid activation membrane penetration activity and its anti-tumor activity by carrying anti-tumor drugs.

[0027] 1. Cellular uptake assay of acid-activated peptides

[0028] HeLa cells were seeded in 24-well plates at 100,000 cells / well and incubated for 24 hours. The original culture medium was then removed from the wells. Serum-free medium containing 5 μM FITC-peptide (pH 7.4 or pH 6.0) was added, and after 1 hour of incubation, cells were collected in 1.5 ml EP tubes, centrifuged at 800 rpm for 5 min, washed with PBS, and resuspended in 500 μL PBS. Flow cytometry was used to analyze peptide entry into the cells. Cells treated with only the culture medium served as a blank control. The ratio of mean fluorescence intensity at pH 6.0 to that at pH 7.4 was calculated. Three independent parallel experiments were performed. Results are shown below. Figure 1 .

[0029] Figure 1 This indicates that, compared with the control, the average fluorescence intensity of LH, LH-K, LH-2K, L5H9 and L5H9K-C6 at pH 6.0 was significantly higher than that at pH 7.4, demonstrating acid-activated transmembrane activity.

[0030] HeLa cells were seeded in small dishes at a density of 60,000 cells / dish. After 24 hours of culture, the cells were incubated for 1 hour with serum-free medium containing 5 μM FITC-peptide at pH 7.4 or 6.0. The cells were then washed once with PBS, with 1 ml of PBS added to each dish. The entry of the peptide into the cells was observed using a laser confocal microscope. Three independent parallel experiments were performed. Results are shown below. Figure 2 As shown.

[0031] from Figure 2 As can be seen, at pH 7.4, LH, LH-K, LH-2K, L5H9, and L5H9K-C6 exhibited almost no transmembrane activity. However, at pH 6.0, all of the above peptides showed significant acid-activated transmembrane activity, and LH-K demonstrated a significantly stronger ability to carry the fluorescent dye FITC into the cell than the other peptides, exhibiting superior acid-activated transmembrane activity.

[0032] 2. In vitro antitumor activity

[0033] HeLa cells were seeded in 96-well plates at 5000 cells / well and incubated for 24 h. Cells were then incubated for 1 h with serum-free culture medium (pH 7.4 or pH 6.0) containing different concentrations of CPT or CPT-peptide. The original culture medium was aspirated, and 100 μL of culture medium containing 10% serum was added to each well. Cells were cultured for another 72 h, and then 10 μL of MTT was added to each well. Cells were incubated for 4 h, and then the liquid in the wells was aspirated. 150 μL of DMSO was added to each well, and the cell plate was shaken on a microplate reader for 5 min. The absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated. Wells containing only culture medium served as blank controls. Three independent parallel experiments were performed. Results are shown below. Figure 3 As shown.

[0034] from Figure 3 As can be seen, compared with CPT (camptothecin), CPT-LH, CPT-LH-K, CPT-LH-2K, CPT-L5H9, and CPT-L5H9K-C6 all exhibited acid-activated antitumor activity. They showed superior antitumor activity at pH 6.0. Similar results were obtained by performing the same procedure on MCF-7 cells, as shown in the table below. Figure 4 This further illustrates that LH, LH-K, LH-2K, L5H9, and L5H9K-C6 possess superior acid-activated transmembrane activity, enabling them to carry more CPT into the cell under pH 6.0 conditions and exert acid-responsive antitumor activity.

[0035] 3. Toxicity test

[0036] (1) 24h-MTT experiment

[0037] HeLa cells were seeded in 96-well plates at 5000 cells / well and cultured for 24 h. Then, the cells were incubated for 24 h with culture medium containing 5% serum and different concentrations of peptide carriers (pH 7.4). 10 μL of MTT was added to each well, and incubation continued for 4 h. The liquid in the wells was aspirated, and 150 μL of DMSO was added to each well. The plates were shaken on a microplate reader for 5 min, and the absorbance at 490 nm was measured using a microplate reader to calculate cell viability. Wells containing only culture medium served as blank controls. Three independent parallel experiments were performed. Results are shown below. Figure 5 As shown.

[0038] Figure 5 The results showed that LH, LH-K, LH-2K, L5H9, and L5H9K-C6 exhibited low cytotoxicity against HeLa cells at pH 7.4. Even at the maximum concentration of 40 μM, cell viability remained above 70% 24 h after drug treatment. Similar results were obtained using the same method on MCF-7 cells; LH, LH-K, LH-2K, L5H9, and L5H9K-C6 showed low cytotoxicity against MCF-7 cells. (See attached results). Figure 6 .

[0039] (2) Hemolysis test

[0040] Fresh mouse blood was collected, centrifuged at 800 rpm for 5 min, and the supernatant was discarded. Red blood cells were washed three times with PBS. PBS solution containing 8% red blood cells was seeded into 96-well plates at 100 μL / well. Different concentrations of peptide carrier solutions prepared with PBS were added to the plates containing red blood cells at 100 μL / well. The cell plates were shaken on a microplate for 1 min, incubated at 37°C for 1 h, centrifuged at 1200 g for 15 min, and 100 μL of supernatant was transferred from each well to a new 96-well plate. The absorbance of the supernatant at 490 nm was measured using a microplate reader. The hemolysis rate of the peptide carrier was calculated. Wells treated with 2% Triton X-100 served as positive controls, and wells treated with PBS served as negative controls. Three independent parallel experiments were performed. Results are shown below. Figure 7 As shown.

[0041] from Figure 7 It can be seen that the acid-activated transmembrane peptide carriers LH, LH-K, LH-2K, L5H9 and L5H9K-C6 still have almost zero hemolysis rate even at the maximum concentration of 200 μM, and show almost no hemolytic toxicity.

[0042] The advantages of this invention compared to the prior art are as follows:

[0043] 1. The acid-activated transmembrane polypeptide carrier of the present invention is rich in leucine, histidine and an appropriate amount of positively charged amino acid X in its structure, and its general structural formula is: L a H b X n This type of polypeptide carrier has a simple structure and does not limit the sequence of the three amino acids.

[0044] 2. The acid-activated transmembrane polypeptide carrier of this invention maximizes the acid responsiveness of the polypeptide carrier while reducing its toxicity through charge and hydrophobicity optimization. Studies show that the acid-activated transmembrane polypeptide carrier of this invention exhibits superior acid-activated transmembrane activity and can effectively carry the antitumor drug CPT into cells at pH 6.0, demonstrating superior acid-activated antitumor activity. Furthermore, the acid-activated transmembrane polypeptide designed in this invention exhibits low cytotoxicity and hemolytic toxicity. In addition, the rapid transmembrane permeation activity exhibited by this peptide after carrying CPT makes it a promising candidate for the preparation of antitumor drugs and the treatment of multidrug resistance. Attached Figure Description

[0045] Figure 1 This is a flow cytometry experiment showing the ratio of average fluorescence intensity of the peptide carrier at pH 6.0 and pH 7.4.

[0046] Figure 2 This is a laser confocal microscope image showing a polypeptide carrier carrying FITC into the cell under different pH conditions.

[0047] Figure 3 This is a graph showing the in vitro antitumor activity of CPT linked to a polypeptide carrier on HeLa cells.

[0048] Figure 4 This is a graph showing the in vitro antitumor activity of CPT linked to a polypeptide carrier on MCF-7 cells.

[0049] Figure 5 This is a graph showing the MTT assay results of the polypeptide carrier acting on HeLa cells for 24 hours at pH 7.4.

[0050] Figure 6 This is a graph showing the MTT assay results of the polypeptide carrier acting on MCF-7 cells for 24 hours at pH 7.4.

[0051] Figure 7 This is a diagram showing the hemolysis results of the polypeptide carrier;

[0052] Figure 8 This is the mass spectrum of LH;

[0053] Figure 9 This is a FITC-LH mass spectrum;

[0054] Figure 10 This is a mass spectrum of Cys-LH.

[0055] Figure 11 It is the mass spectrum of 2-(2-pyridinyldithio)-ethanol;

[0056] Figure 12 This is the mass spectrum of activated CPT;

[0057] Figure 13 This is a CPT-LH mass spectrum;

[0058] Figure 14 This is the mass spectrum of LH-K;

[0059] Figure 15 It is a FITC-LH-K mass spectrum;

[0060] Figure 16 This is a mass spectrum of Cys-LH-K;

[0061] Figure 17 This is the mass spectrum of CPT-LH-K;

[0062] Figure 18 This is the mass spectrum of LH-2K;

[0063] Figure 19 This is the mass spectrum of FITC-LH-2K;

[0064] Figure 20 This is the mass spectrum of Cys-LH-2K;

[0065] Figure 21 This is the mass spectrum of CPT-LH-2K;

[0066] Figure 22 This is the mass spectrum of L5H9;

[0067] Figure 23 This is the mass spectrum of FITC-L5H9;

[0068] Figure 24 This is the mass spectrum of Cys-L5H9;

[0069] Figure 25 This is the mass spectrum of CPT-L5H9;

[0070] Figure 26 It is the mass spectrum of L5H9K-C6;

[0071] Figure 27 This is the mass spectrum of FITC-L5H9K-C6;

[0072] Figure 28 It is the mass spectrum of Cys-L5H9K-C6;

[0073] Figure 29 It is a mass spectrum of CPT-L5H9K-C6. Detailed Implementation

[0074] The synthesis process of the polypeptide carrier with acid-activated membrane-penetrating activity of the present invention will be further described below through specific embodiments.

[0075] Example 1: Synthesis of LH and FITC / CPT-LH

[0076] Part 1: Synthesis of LH

[0077] 0.5 g of MBHA resin and 12 ml of DCM were placed in a synthesizer and stirred for 30 min to swell the resin. The resin was washed three times with DMF and analyzed using the ninhydrin colorimetric method. The resin was then washed with a DMF solution containing 20% ​​piperidine to remove the protecting amino groups. Residual piperidine was washed away with DMF, and the resin showed a blue color when analyzed using the ninhydrin colorimetric method. 371.8 mg of Fmoc-His(Trt)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF and mixed with 0.2 ml of DIEA. This mixture was then added to the synthesizer and stirred for 1 h. Unreacted amino acids were washed away with DMF, and the resin showed a colorless color when analyzed using the ninhydrin colorimetric method, yielding Fmoc-His-MBHA. The Fmoc group on histidine was removed using a DMF solution containing 20% ​​piperidine, and the subsequent steps were the same as above until Fmoc-Leu-His-His-Leu-Leu-Leu-His-His-Leu-His-Leu-Leu-His-His-MBHA was synthesized. The Fmoc group on the last Leu was removed using a DMF solution containing 20% ​​piperidine. The peptide chain was cleaved from the resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v). The synthesized peptide was extracted with deionized water and diethyl ether, freeze-dried, and the crude peptide was obtained. After HPLC purification, the pure peptide LH was obtained. The molecular weight was 1793 Da, and the mass spectrum is shown below. Figure 8 As shown.

[0078] Part Two: Synthesis of FITC-LH

[0079] 0.5 g of resin was swollen and stirred in DCM for 30 min. The resin was washed with DMF solution containing 20% ​​piperidine to remove the protecting groups of amino groups on the resin. Residual piperidine was washed away with DMF. 371.8 mg of Fmoc-His(Trt)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was then added to a peptide synthesizer and stirred for 1 h. Residual amino acids were washed away with DMF. The resin was colorless as determined by the ninhydrin colorimetric method, yielding Fmoc-His-MBHA. The resin was washed with DMF solution containing 20% ​​piperidine to remove the Fmoc groups on histidine residues. Subsequent steps were repeated until Fmoc-Leu-His-His-Leu-Leu-Leu-His-His-His-Leu-Leu-His-MBHA was synthesized. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc group from leucine. 117 mg of FITC was dissolved in DMF, and then 0.4 ml of DIEA was added. The mixture was then added to a peptide synthesizer and stirred overnight in the dark. Residual FITC was washed away with DMF, and the resin was confirmed to be yellow by ninhydrin staining, yielding FITC-Leu-His-His-Leu-Leu-His-His-His-His-Leu-Leu-His-His-MBHA. The peptide chain was cleaved from the resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v). The synthesized peptide was extracted with deionized water and diethyl ether, freeze-dried, and the crude peptide was obtained. After HPLC purification, the pure peptide FITC-LH was obtained. The molecular weight was 2295 Da, and the mass spectrum is shown below. Figure 9 As shown.

[0080] Part 3: Synthesis of CPT-LH

[0081] (1) Synthesis of Cys-LH

[0082] 0.5 g of resin was swollen with DCM and stirred in a peptide synthesizer for 30 min. The resin was washed three times with DMF. The resin was then washed four times with a DMF solution containing 20% ​​piperidine to remove the protecting groups of the amino groups on the resin. Residual piperidine was washed away with DMF. 371.8 mg of Fmoc-His(Trt)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was then added to the peptide synthesizer and stirred for 1 h. Unreacted amino acids were washed away with DMF. The coupling of amino acids was determined by the ninhydrin colorimetric method. If the resin was colorless, the amino acid coupling was successful, yielding Fmoc-His-MBHA. The Fmoc group on histidine was removed using a DMF solution containing 20% ​​piperidine, followed by the same steps until Fmoc-Cys-Leu-His-His-Leu-Leu-Leu-His-His-Leu-Leu-His-His-MBHA was synthesized. The Fmoc group on cysteine ​​was removed using a DMF solution containing 20% ​​piperidine. The peptide chain was cleaved from the MBHA resin using a cleaving agent (TFA: Triisopropylsilane: 1,2-Ethanedithiol: H2O = 9.4:0.25:0.25:1v:v:v):v. Extraction was performed with ether and water to obtain an aqueous solution of the peptide, which was then freeze-dried. The pure peptide Cys-LH was obtained by HPLC purification. The molecular weight was 1896 Da, and the mass spectrum is shown below. Figure 10 As shown.

[0083] (2) Synthesis of 2-(2-pyridinyldithio)-ethanol

[0084] 330 mg of 2,2-dithiopyridine was added to a 50 mL round-bottom flask. A magnetic stir bar was placed inside the flask, and the flask was sealed. The inside of the flask was evacuated and purged with argon gas. 5 mL of methanol was added to the flask and stirred to dissolve. 87 mg of mercaptoethanol was dissolved in 5 mL of methanol and then added dropwise to the round-bottom flask. The reaction was stirred for 3 h. The reaction product was purified by silica gel column chromatography to obtain 2-(2-pyridinyldithio)-ethanol with a molecular weight of 187 Da. The mass spectrum is shown below. Figure 11 As shown.

[0085] (3) Activation of CPT

[0086] 372.5 mg CPT and 356.6 mg DMAP were placed in a 25 mL round-bottom flask, a magnetic stir bar was added, the flask was evacuated, and argon gas was introduced. 5 mL of DCM was added, and the mixture was stirred in an ice bath for 15 min. Triphosgene was dissolved in 3 mL of DCM and added dropwise to the round-bottom flask. The reaction was continued in an ice bath for 15 min, then the ice bath was removed. The previously obtained 2-(2-pyridinyldithio)-ethanol was dissolved in 3 mL of DCM and added dropwise to the flask. The mixture was reacted overnight in the dark. The reaction product was purified by silica gel column chromatography. The molecular weight was 562 Da, and the mass spectrum is shown below. Figure 12 As shown.

[0087] (4) Synthesis of CPT-LH

[0088] A magnetic stir bar was placed in a 5 ml round-bottom flask, sealed, evacuated, and filled with argon gas. 14.5 mg of Cys-LH and 6.5 mg of activated CPT were dissolved separately in 1 ml of DMSO and added to the round-bottom flask. The mixture was stirred and reacted overnight. CPT-LH was obtained by HPLC purification, with a molecular weight of 2346 Da. The mass spectrum is shown below. Figure 13 As shown.

[0089] Example 2: Synthesis of LH-K and FITC / CPT-LH-K

[0090] Part 1: Synthesis of LH-K

[0091] 0.5 g of MBHA resin was stirred and swollen with DCM for 30 min, and then the DCM was washed away with DMF. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the protecting group of the amino group, and residual piperidine was washed away with DMF. The resin showed a blue color when tested with ninhydrin. 281.2 mg of Fmoc-Lys(Boc)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was stirred and added to a synthesizer, and the reaction was carried out for 1 h. Unreacted amino acids were washed away with DMF, and the resin showed a colorless color when tested with ninhydrin, yielding Fmoc-Lys-MBHA. The resin was washed with DMF containing 20% ​​piperidine to remove the Fmoc group on the lysine residues. Subsequent steps were repeated until Fmoc-Leu-His-His-Leu-Leu-His-His-His-Leu-Leu-His-His-Lys-MBHA was synthesized. The resin was then washed with DMF solution containing 20% ​​piperidine to remove the Fmoc group on the last leucine residue. The resin showed a blue color when tested with ninhydrin. The peptide chain was then cleaved from the MBHA resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v). The extract was obtained by extraction with ether and water, yielding an aqueous solution of the peptide, which was then freeze-dried. The pure peptide LH-K was obtained by HPLC purification. The molecular weight was 1921 Da, and the mass spectrum is shown below. Figure 14 As shown.

[0092] Part Two: Synthesis of FITC-LH-K

[0093] 0.5 g of MBHA resin was swollen and stirred for 30 min using DCM. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the protecting groups of the amino groups on the resin, and residual piperidine was washed away with DMF. 281.2 mg of Fmoc-Lys(Boc)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was then added to a peptide synthesizer and stirred for 1 h. Residual amino acids were washed away with DMF, and the resin was colorless as determined by a ninhydrin colorimetric reaction, yielding Fmoc-Lys-MBHA. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc groups on the lysine residues. Subsequent steps were repeated until Fmoc-Leu-His-His-Leu-Leu-Leu-His-His-His-His-Lys-MBHA was obtained. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc group from leucine. 117 mg of FITC was dissolved in DMF, and 0.4 ml of DIEA was added. The mixture was then added to a peptide synthesizer and stirred overnight in the dark. Residual FITC was washed away with DMF, and the resin was confirmed to be yellow by ninhydrin staining, yielding FITC-Leu-His-His-Leu-Leu-His-His-His-His-Leu-Leu-His-His-Lys-MBHA. The peptide chain was then cleaved from the MBHA resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v). Extraction with ether and water yielded an aqueous solution of the peptide, which was then freeze-dried. The purified peptide FITC-LH-K was obtained by HPLC purification. The molecular weight was 2423 Da, and the mass spectrum is shown below. Figure 15 As shown.

[0094] Part 3: Synthesis of CPT-LH-K

[0095] (1) Synthesis of Cys-LH-K

[0096] 0.5 g of MBHA resin was swollen and stirred in DCM for 30 min. The resin was washed three times with DMF. The protecting group of the amino group was removed with a DMF solution containing 20% ​​piperidine, and the residual piperidine was washed away with DMF. 281.2 mg of Fmoc-Lys(Boc)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was then added to the synthesizer and stirred for 1 h. The resin was colorless as determined by the ninhydrin colorimetric method, yielding Fmoc-Lys-MBHA. The resin was washed four times with a DMF solution containing 20% ​​piperidine to remove the Fmoc group on the lysine residue. Subsequent steps were repeated until Fmoc-Cys-Leu-His-His-His-Leu-Leu-His-His-His-His-His-Lys-MBHA was synthesized. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc group from the cysteine ​​residues. The peptide chain was cleaved from the MBHA resin using a cleaving agent (TFA: Triisopropylsilane: 1,2-Ethanedithiol: H2O = 9.4:0.25:0.25:1v:v:v:v). The extract was then obtained by extraction with ether and water, yielding an aqueous solution of the peptide, which was freeze-dried. The pure peptide Cys-LH-K was obtained by HPLC purification. The molecular weight is 2024 Da, and the mass spectrum is shown below. Figure 16 As shown.

[0097] (2) Synthesis of 2-(2-pyridinyldithio)-ethanol

[0098] Same as Example 1

[0099] (3) Activation of CPT

[0100] Same as Example 1

[0101] (4) Synthesis of CPT-LH-K

[0102] A magnetic stir bar was placed in a 5 ml round-bottom flask, sealed, evacuated, and purged with argon gas. 15.6 mg of Cys-LH-K and 6.5 mg of activated CPT were dissolved separately in 1 ml of DMSO and added to the round-bottom flask. The mixture was stirred and reacted overnight. The product CPT-LH-K was obtained by HPLC purification. The molecular weight was 2474 Da. The mass spectrum is shown below. Figure 17 As shown.

[0103] Example 3: Synthesis of LH-2K and FITC / CPT-LH-2K

[0104] Part 1: Synthesis of LH-2K

[0105] 0.5 g of MBHA resin was stirred and swollen with DCM for 30 min, and then the DCM was washed away with DMF. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the protecting group of the amino group, and residual piperidine was washed away with DMF. The resin showed a blue color when tested with ninhydrin. 281.2 mg of Fmoc-Lys(Boc)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was stirred and added to a synthesizer, and the reaction was carried out for 1 h. Unreacted amino acids were washed away with DMF, and the resin showed a colorless color when tested with ninhydrin, yielding Fmoc-Lys-MBHA. The resin was washed with DMF containing 20% ​​piperidine to remove the Fmoc group on the lysine residues. Subsequent steps were repeated until Fmoc-Leu-His-His-Leu-Leu-His-His-His-Leu-His-His-Lys-Lys-MBHA was synthesized. The resin was then washed with DMF solution containing 20% ​​piperidine to remove the Fmoc group on the last leucine residue. The resin showed a blue color when tested with ninhydrin. The peptide chain was then cleaved from the MBHA resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v). The extract was obtained by extraction with ether and water, yielding an aqueous solution of the peptide, which was then freeze-dried. The pure peptide LH-2K was obtained by HPLC purification. The molecular weight was 2049 Da, and the mass spectrum is shown below. Figure 18 As shown.

[0106] Part Two: Synthesis of FITC-LH-2K

[0107] 0.5 g of MBHA resin was swollen and stirred for 30 min using DCM. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the protecting groups of the amino groups on the resin, and residual piperidine was washed away with DMF. 281.2 mg of Fmoc-Lys(Boc)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was then added to a peptide synthesizer and stirred for 1 h. Residual amino acids were washed away with DMF, and the resin was colorless as determined by a ninhydrin colorimetric reaction, yielding Fmoc-Lys-MBHA. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc groups on the lysine residues. Subsequent steps were repeated until Fmoc-Leu-His-His-Leu-Leu-Leu-His-His-His-His-Lys-Lys-MBHA was synthesized. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc group on leucine. 117 mg of FITC was dissolved in DMF, and 0.4 ml of DIEA was added. The mixture was then added to a peptide synthesizer and stirred overnight in the dark. Residual FITC was washed away with DMF, and the resin was confirmed to be yellow by ninhydrin staining, yielding FITC-Leu-His-His-Leu-Leu-His-His-His-His-Leu-His-His-Lys-Lys-MBHA. The peptide chain was then cleaved from the MBHA resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v). Extraction with ether and water yielded an aqueous solution of the peptide, which was then freeze-dried. The purified peptide FITC-LH-2K was obtained by HPLC purification. The molecular weight was 2551 Da, and the mass spectrum is shown below. Figure 19 As shown.

[0108] Part 3: Synthesis of CPT-LH-2K

[0109] (1) Synthesis of Cys-LH-2K

[0110] 0.5 g of MBHA resin was swollen and stirred in DCM for 30 min. The resin was washed three times with DMF. The protecting group of the amino group was removed with a DMF solution containing 20% ​​piperidine, and the residual piperidine was washed away with DMF. 281.2 mg of Fmoc-Lys(Boc)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was then added to the synthesizer and stirred for 1 h. The resin was colorless as determined by the ninhydrin colorimetric method, yielding Fmoc-Lys-MBHA. The resin was washed four times with a DMF solution containing 20% ​​piperidine to remove the Fmoc group on the lysine residues. Subsequent steps were repeated until Fmoc-Cys-Leu-His-His-His-Leu-Leu-His-His-His-His-His-Lys-Lys-MBHA was synthesized. The resin was then washed again with a DMF solution containing 20% ​​piperidine to remove the Fmoc group on the cysteine ​​residues. The peptide chain was cleaved from the MBHA resin using a cleaving agent (TFA: Triisopropylsilane: 1,2-Ethanedithiol: H2O = 9.4:0.25:0.25:1v:v:v):v. The peptide was extracted with ether and water to obtain an aqueous solution, which was then freeze-dried. The purified peptide Cys-LH-2K was obtained by HPLC purification. The molecular weight was 2152 Da. The mass spectrum is shown below. Figure 20 As shown.

[0111] (2) Synthesis of 2-(2-pyridinyldithio)-ethanol

[0112] Same as Example 1

[0113] (3) Activation of CPT

[0114] Same as Example 1

[0115] (4) Synthesis of CPT-LH-2K

[0116] A magnetic stir bar was placed in a 5 ml round-bottom flask, sealed, evacuated, and purged with argon gas. 10.1 mg of Cys-LH-2K and 4 mg of activated CPT were dissolved separately in 1 ml of DMSO and added to the round-bottom flask. The mixture was stirred and reacted overnight. The product CPT-LH-2K was obtained by HPLC purification. The molecular weight was 2602 Da. The mass spectrum is shown below. Figure 21 As shown.

[0117] Example 4: Synthesis of L5H9 and FITC / CPT-L5H9

[0118] Part 1: Synthesis of L5H9

[0119] 0.5 g of MBHA resin and 12 ml of DCM were placed in a synthesizer and stirred for 30 min to swell the resin. The resin was washed three times with DMF and analyzed using the ninhydrin colorimetric method. The resin was then washed with a DMF solution containing 20% ​​piperidine to remove the protecting amino groups. Residual piperidine was washed away with DMF, and the resin showed a blue color when analyzed using the ninhydrin colorimetric method. 371.8 mg of Fmoc-His(Trt)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF and mixed with 0.2 ml of DIEA. This mixture was then added to the synthesizer and stirred for 1 h. Unreacted amino acids were washed away with DMF, and the resin showed a colorless color when analyzed using the ninhydrin colorimetric method, yielding Fmoc-His-MBHA. The Fmoc group on histidine was removed using a DMF solution containing 20% ​​piperidine, and the subsequent steps were the same as above until Fmoc-Leu-His-His-Leu-His-His-His-Leu-Leu-His-His-His-His-MBHA was synthesized. The last Fmoc group of the Leu group was removed using a DMF solution containing 20% ​​piperidine. The peptide chain was cleaved from the resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v). The synthesized peptide was extracted with deionized water and diethyl ether, freeze-dried, and the crude peptide was obtained. After HPLC purification, the pure peptide L5H9 was obtained. The molecular weight was 1817 Da, and the mass spectrum is shown below. Figure 22 As shown.

[0120] Part Two: Synthesis of FITC-L5H9

[0121] 0.5 g of resin was swollen and stirred in DCM for 30 min. The resin was washed with DMF solution containing 20% ​​piperidine to remove the protecting groups of the amino groups on the resin. Residual piperidine was washed away with DMF. 371.8 mg of Fmoc-His(Trt)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was then added to a peptide synthesizer and stirred for 1 h. Residual amino acids were washed away with DMF. The resin was colorless as determined by the ninhydrin colorimetric method, yielding Fmoc-His-MBHA. The resin was washed with DMF solution containing 20% ​​piperidine to remove the Fmoc groups on the histidine residues. Subsequent steps were repeated until Fmoc-Leu-His-His-Leu-His-His-His-His-His-His-His-MBHA was synthesized. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc group from leucine. 117 mg of FITC was dissolved in DMF, and then 0.4 ml of DIEA was added. The mixture was then added to a peptide synthesizer and stirred overnight in the dark. Residual FITC was washed away with DMF, and the resin was tested for yellow color using the ninhydrin method, yielding FITC-Leu-His-His-Leu-His-His-His-His-Leu-His-His-His-MBHA. The peptide chain was cleaved from the resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v). The synthesized peptide was extracted with deionized water and diethyl ether, freeze-dried, and the crude peptide was obtained. After HPLC purification, the pure peptide FITC-L5H9 was obtained. The molecular weight was 2319 Da, and the mass spectrum is shown below. Figure 23 As shown.

[0122] Part 3: Synthesis of CPT-L5H9

[0123] (1) Synthesis of Cys-L5H9

[0124] 0.5 g of resin was swollen with DCM and stirred in a peptide synthesizer for 30 min. The resin was washed three times with DMF. The resin was then washed four times with a DMF solution containing 20% ​​piperidine to remove the protecting groups of the amino groups on the resin. Residual piperidine was washed away with DMF. 371.8 mg of Fmoc-His(Trt)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was then added to the peptide synthesizer and stirred for 1 h. Unreacted amino acids were washed away with DMF. The coupling of amino acids was determined by the ninhydrin colorimetric method. If the resin was colorless, the amino acid coupling was successful, yielding Fmoc-His-MBHA. The Fmoc group on histidine was removed using a DMF solution containing 20% ​​piperidine, followed by the same steps until Fmoc-Cys-Leu-His-His-His-His-His-His-His-His-His-His-His-MBHA was synthesized. The Fmoc group on cysteine ​​was removed using a DMF solution containing 20% ​​piperidine. The peptide chain was cleaved from the MBHA resin using a cleaving agent (TFA: Triisopropylsilane: 1,2-Ethanedithiol: H2O = 9.4:0.25:0.25:1v:v:v):v. Extraction was performed with ether and water to obtain an aqueous solution of the peptide, which was then freeze-dried. The pure peptide Cys-L5H9 was obtained by HPLC purification. The molecular weight was 1920 Da, and the mass spectrum is shown below. Figure 24 As shown.

[0125] (2) Synthesis of 2-(2-pyridinyldithio)-ethanol

[0126] Same as Example 1

[0127] (3) Activation of CPT

[0128] Same as Example 1

[0129] (4) Synthesis of CPT-L5H9

[0130] A magnetic stir bar was placed in a 5 ml round-bottom flask, sealed, evacuated, and filled with argon gas. 15 mg of Cys-L5H9 and 6.7 mg of activated CPT were dissolved separately in 1 ml of DMSO and added to the round-bottom flask. The mixture was stirred and reacted overnight. CPT-L5H9 was obtained by HPLC purification, with a molecular weight of 2370 Da. The mass spectrum is shown below. Figure 25 As shown.

[0131] Example 5: Synthesis of L5H9K-C6 and FITC / CPT-L5H9K-C6

[0132] Part 1: Synthesis of L5H9K-C6

[0133] 0.5 g of MBHA resin was stirred and swollen with DCM for 30 min, and then the DCM was washed away with DMF. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the protecting groups of the amino groups, and residual piperidine was washed away with DMF. The resin showed a blue color when tested with ninhydrin. 384.5 mg of Fmoc-Lys(mtt)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was stirred and added to a synthesizer, and the reaction was carried out for 1 h. Unreacted amino acids were washed away with DMF, and the resin showed a colorless color when tested with ninhydrin, yielding Fmoc-Lys(mtt)-MBHA. The resin was washed with DMF containing 20% ​​piperidine to remove the Fmoc group on the lysine residue. Subsequent steps were repeated until Fmoc-Leu-His-His-Leu-His-His-His-Leu-His-His-His-His-Lys(mtt)-MBHA was synthesized. The resin was washed with dichloromethane solution containing 1% TFA to remove the mtt group from the Lys side chain. The resin showed a blue color when tested with ninhydrin. 69.7 mg of C6H was dissolved in DMF. 12 After adding O2, 81.2 mg HOBT, and 227.6 mg HBTU, 0.2 ml DIEA was added, mixed well, and added to a synthesizer. The mixture was stirred for 1 h. Unreacted hexanoic acid was washed away with DMF, and the resin was colorless as determined by ninhydrin colorimetry, yielding Fmoc-Leu-His-His-Leu-His-His-His-His-His-His-His-His-Lys(C6)-MBHA. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the last leucine Fmoc group. The resin was blue as determined by ninhydrin colorimetry. The peptide chain was then cleaved from the MBHA resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v), extracted with ether and water to obtain an aqueous solution of the peptide, which was then freeze-dried. The peptide L5H9K-C6 was purified by HPLC. Its molecular weight is 2043 Da, and its mass spectrum is shown below. Figure 26 As shown.

[0134] Part Two: Synthesis of FITC-L5H9K-C6

[0135] 0.5 g of MBHA resin was stirred and swollen with DCM for 30 min, and then the DCM was washed away with DMF. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the protecting groups of the amino groups, and residual piperidine was washed away with DMF. The resin showed a blue color when tested with ninhydrin. 384.5 mg of Fmoc-Lys(mtt)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was stirred and added to a synthesizer, and the reaction was carried out for 1 h. Unreacted amino acids were washed away with DMF, and the resin showed a colorless color when tested with ninhydrin, yielding Fmoc-Lys(mtt)-MBHA. The resin was washed with DMF containing 20% ​​piperidine to remove the Fmoc group on the lysine residue. Subsequent steps were repeated until Fmoc-Leu-His-His-Leu-His-His-His-Leu-His-His-His-His-Lys(mtt)-MBHA was synthesized. The resin was washed with dichloromethane solution containing 1% TFA to remove the mtt group from the Lys side chain. The resin showed a blue color when tested with ninhydrin. 69.7 mg of C6H was dissolved in DMF. 12 After adding O2, 81.2 mg of HOBT, and 227.6 mg of HBTU, 0.2 ml of DIEA was added, mixed well, and then added to the synthesizer. The mixture was stirred for 1 h. Unreacted hexanoic acid was washed away with DMF, and the resin was tested with ninhydrin colorimetric reaction; the result was colorless, yielding Fmoc-Leu-His-His-Leu-His-His-His-His-His-His-His-His-Lys(C6)-MBHA. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the Fmoc group from the leucine. 117 mg of FITC was dissolved in DMF, and then 0.4 ml of DIEA was added. The mixture was added to the peptide synthesizer and stirred overnight in the dark. Residual FITC was washed away with DMF, and the resin tested yellow using the ninhydrin colorimetric method, yielding FITC-Leu-His-His-Leu-His-His-His-His-His-His-His-Lys(C6)-MBHA. The peptide chain was then cleaved from the MBHA resin using a cleaving agent (TFA:Triisopropylsilane:H2O = 9.5:0.25:0.25v:v:v). Extraction with ether and water yielded an aqueous solution of the peptide, which was then freeze-dried. Purification by HPLC yielded the pure peptide FITC-L5H9K-C6 with a molecular weight of 2545 Da. The mass spectrum is shown below. Figure 27 As shown.

[0136] Part 3: Synthesis of CPT-L5H9K-C6

[0137] (1) Synthesis of Cys-L5H9K-C6

[0138] 0.5 g of MBHA resin was stirred and swollen with DCM for 30 min, and then the DCM was washed away with DMF. The resin was washed with a DMF solution containing 20% ​​piperidine to remove the protecting groups of the amino groups, and residual piperidine was washed away with DMF. The resin showed a blue color when tested with ninhydrin. 384.5 mg of Fmoc-Lys(mtt)-OH, 81.2 mg of HOBT, and 227.6 mg of HBTU were dissolved in DMF, and 0.2 ml of DIEA was added. The mixture was stirred and added to a synthesizer, and the reaction was carried out for 1 h. Unreacted amino acids were washed away with DMF, and the resin showed a colorless color when tested with ninhydrin, yielding Fmoc-Lys(mtt)-MBHA. The resin was washed with DMF containing 20% ​​piperidine to remove the Fmoc group on the lysine residue. Subsequent steps were repeated until Fmoc-Cys-Leu-His-His-Leu-His-His-His-His-His-His-His-His-Lys(mtt)-MBHA was synthesized. The resin was washed with dichloromethane solution containing 1% TFA to remove the mtt group from the Lys side chain. The resin was tested for blue color using the ninhydrin colorimetric method. 69.7 mg of C6H was dissolved in DMF. 12 After adding O2, 81.2 mg HOBT, and 227.6 mg HBTU, 0.2 ml DIEA was added, mixed well, and then added to a synthesizer. The mixture was stirred for 1 h. Unreacted hexanoic acid was washed away with DMF. The resin was tested with ninhydrin colorimetric assay; the result was colorless, yielding Fmoc-Cys-Leu-His-His-His-His-His-His-His-His-His-His-His-His-His-Lys(C6)-MBHA. The resin was washed with DMF solution containing 20% ​​piperidine to remove the last cysteine ​​Fmoc group. The resin was tested with ninhydrin colorimetric assay; the result was blue. The peptide chain was then cleaved from the MBHA resin using a cleaving agent (TFA: Triisopropylsilane: 1,2-Ethanedithiol: H2O = 9.4:0.25:0.25:1v:v:v:v). Extraction with ether and water yielded an aqueous solution of the peptide, which was then freeze-dried. Purification by HPLC yielded pure peptide Cys-L5H9K-C6 with a molecular weight of 2146 Da. The mass spectrum is shown below. Figure 28 As shown.

[0139] (2) Synthesis of 2-(2-pyridinyldithio)-ethanol

[0140] Same as Example 1

[0141] (3) Activation of CPT

[0142] Same as Example 1

[0143] (4) Synthesis of CPT-L5H9K-C6

[0144] A magnetic stir bar was placed in a 5 ml round-bottom flask, sealed, evacuated, and filled with argon gas. 10 mg of Cys-L5H9K-C6 and 4.2 mg of activated CPT were dissolved separately in 1 ml of DMSO and added to the round-bottom flask. The mixture was stirred and reacted overnight. CPT-L5H9K-C6 was obtained by HPLC purification, with a molecular weight of 2596 Da. The mass spectrum is shown below. Figure 29 As shown. sequence list <110> Ni Jingman Wang Rui <120> An acid-activated membrane-penetrating polypeptide carrier and its application <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <400> 1 Leu His His Leu Leu His His Leu His His Leu Leu His His 1 5 10 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <400> 2 Leu His His Leu Leu His His Leu His His Leu Leu His His Lys 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <400> 3 Leu His His Leu Leu His His Leu His His Leu Leu His His Lys Lys 1 5 10 15 <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <400> 4 Leu His His Leu His His Leu Leu His His Leu His His His 1 5 10 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <400> 5 Leu His His Leu His His Leu Leu His His Leu His His His Lys 1 5 10 15

Claims

1. An acid-activated membrane-penetrating polypeptide carrier, characterized in that, The polypeptide carrier is: L6H8K1, abbreviated as LH-K, has the amino acid sequence shown in SEQ ID No. 2; Alternatively: L6H8K2, abbreviated as LH-2K, its amino acid sequence is shown in SEQ ID No. 3; Alternatively: L5H9K0, abbreviated as L5H9, its amino acid sequence is shown in SEQ ID No. 4; Alternatively: L5H9(K-C6)1, abbreviated as (L5H9K-C6), whose amino acid sequence is shown in SEQ ID No.5; wherein L5H9(K-C6)1 is obtained by modifying the carbon terminus of peptide chain L5H9K0 with hexanoic acid.

2. The application of the acid-activated membrane-penetrating polypeptide carrier as described in claim 1 in the preparation of targeted carriers or drug delivery systems.

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