Stimuli-responsive polymer-drug conjugates for antitumor drug delivery



Polymer-drug conjugates (polymer-drug conjugates, PDCs) have the advantages of good circulation stability and high drug loading capacity, so they have been widely studied.

Stimuli-responsive PDCs (stimuli responsive PDCs, SRPDCs) can respond to some endogenous stimuli (such as acidic pH environment, changed redox environment and up-regulated enzymes) and external stimuli (such as magnetic field, light, temperature and Under the action of ultrasound), the loaded drug is released responsively, so it is called “smart” drug nano-delivery carrier.

In recent years, the research progress of SRPDCs for anti-tumor drug delivery is reviewed, including its design ideas, smart connection keys used and drug release properties, in order to provide reference for related research.

In addition, in order to promote the successful transformation of SRPDCs technology, the author discusses and analyzes the existing problems, difficulties and future research directions in the current research.

Focus: polymer-drug conjugates; stimuli-responsive drug release; anticancer drug delivery; tumor targeting; nanocarriers

Cancer is a major disease that seriously affects human health and threatens the safety of human life. Chemotherapy is one of the important treatment methods for cancer, especially the main treatment for advanced cancer.

However, many chemotherapeutic drugs have problems such as poor water solubility, poor in vivo distribution selectivity, and high toxicity to normal tissues. Therefore, the development of highly efficient and low-toxicity carriers is of great significance for improving the therapeutic effect of anticancer drugs and reducing their toxic and side effects.

Different from the traditional physical encapsulation method of drug loading, PDCs refer to macromolecular prodrugs prepared by linking drugs with water-soluble polymer carriers through covalent bonds. The model was proposed by Ringsdorf in the mid-1970s.

PDCs generally self-assemble into micelles or nanoparticles in aqueous media, so they can target tumor tissues through enhanced permeability and retention effect (EPR), and enter into them efficiently through endocytic mechanism. cells, thereby bypassing the efflux proteins on the cell membrane and overcoming the advantages of tumor multidrug resistance.

In addition, compared with physically drug-loaded nano-formulations, PDCs have the advantages of high drug-loading capacity and low risk of drug burst release during in vitro transport.

Although many PDCs for tumor therapy have entered the clinical research stage, with the progress of clinical trials, some promising candidate conjugates in previous studies have been terminated due to undesired drug release behavior.

During the transport process, premature or too fast drug release will lead to the loss of PDCs pharmacokinetic advantages or increased toxicity to normal tissues; and too late or too slow drug release after reaching the target site will reduce the therapeutic effect of the drug .

Therefore, the drug release behavior of PDCs (rationality, timeliness, and specificity in time and space), which is mainly determined by the nature of the linkage between the drug and the polymer, is a crucial factor affecting its antitumor effect and safety.

1. Stimuli-responsive PDCs (SRPDCs) in tumor sites

Tumor cells have vigorous metabolism, rapid growth, and high demand for energy and oxygen, leading to enhanced glycolysis of tumor cells and a hypoxic state inside the tumor tissue. Features, such as higher internal temperature of tumor tissue, lower pH, higher intracellular glutathione (GSH) level, and overexpression of certain enzymes (such as metal matrix protease, MMP2).

In addition, artificially applying external stimuli to solid tumors, such as magnetic fields, ultrasound, or light, can also create certain characteristics of the tumor microenvironment that are different from normal tissues.

Taking advantage of the difference between the physiological environment of tumor tissue and normal tissue, selecting linkages that are sensitive to drug release in the tumor environment to prepare SRPDCs can promote the specific release of drugs in tumor sites and improve the therapeutic effect, which is of great significance to promote the research and development of PDCs.

Therefore, the author will summarize and summarize the SRPDCs in the tumor environment in recent years, in order to provide a reference for the research on the intelligent targeted delivery system of anti-tumor drugs.

2. SRPDCs for Small Molecule Anticancer Drug Delivery

Small molecule antineoplastic drugs are the most widely used drugs in clinical chemotherapy regimens. However, many of these drugs have poor water solubility, and the current preparations generally contain solubilizers.

In addition, due to the small relative molecular weight, the distribution of this type of drug in the body is non-selective, and there are obvious toxic and side effects on normal tissues and organs, which limits its clinical efficacy.

PDCs based on small-molecule drugs are the earliest researched macromolecular prodrugs. At the same time, based on the tumor environment of such drugs, SRPDCs are currently the most widely studied type of drug delivery system because they can achieve tumor-targeted and specific drug release. .

According to different drug release mechanisms, this type of SRPDCs can be divided into pH-responsive, redox-responsive, enzyme-sensitive, light-sensitive, and multiple-stimuli-responsive, etc.

2.1 pH-sensitive SRPDCs

Studies have found that the metabolism of solid tumor tissue is accelerated and glycolysis is increased, so its internal pH (about 6. 5-7. 2) is lower than that of blood and normal tissues (pH 7. 4); 4. 5-5. 0) and endosomes (pH 5. 5-6. 5) had lower pH.

Selecting pH-sensitive degradable chemically bonded drugs and polymers can endow PDCs with responsive drug release characteristics in a specific pH environment at the tumor site, which can not only avoid the early release of drugs during the circulation process, but also ensure timely drug release after entering the tumor tissue , help to improve drug accumulation and efficacy in tumor sites.

According to the chemical structure of the linker, this type of SRPDCs can be divided into responsive PDCs mediated by hydrazone bonds, acetals, Schiff bases, etc.

2.1.1 Hydrazone-mediated pH-sensitive SRPDCs

The hydrazone linkage is the most widely studied pH-sensitive linker.

The hydrazine group (generally the end of the polymer connection) and the carbonyl group (the end of the drug connection) are essential groups for the preparation of SRPDCs mediated by hydrazone bonds. For drugs and polymers whose chemical structures do not meet the above requirements, the corresponding groups need to be introduced through chemical modification. .

Doxorubicin (DOX) molecules contain active carbonyl groups and do not require additional modification (modifications may affect drug activity). Therefore, hydrazone bond-mediated SRPDCs were first seen in the study of DOX conjugates.

Sirova et al chose N-(2-hydroxypropyl)methacrylamide (HPMA) as the carrier material to prepare hydrazone-linked HPMA-DOX.

The experimental results showed that compared with free DOX, after preparation into HPMA-DOX, the circulation time of the drug in the body was prolonged, and the distribution in the tumor site was increased; in vivo pharmacodynamic experiments showed that HPMA-DOX could significantly prolong the survival of tumor-bearing mice time, and no adverse reactions of myelosuppression were found.

2.1.2 Other chemical bond-mediated pH-sensitive SRPDCs

In addition to the hydrazone bond, choosing acetal, cis-acotinyl, Schiff base, and β-thiopropionate as the linker can also make PDCs have the function of pH-responsive drug release.

Yang et al prepared a pH-sensitive PEG-b-PC-DOX conjugate using the Schiff base as the linker. The cumulative drug release of the conjugate was greater than 50% in 24 h at pH 5.0; while at pH 7 . 4 conditions, drug release is only about 23%.

In addition, PEG-b-PC-DOX conjugate micelles had a stronger growth inhibitory effect on drug-resistant MCF-7/ADR cells compared to DOX solution, which may be due to passive diffusion transmembrane transport with free drug Differently, nanosized PDCs can bypass multidrug resistance-associated p-gp efflux proteins and release drugs rapidly and responsively in the acidic environment of lysosomes.

SRPDCs based on the acidic environment of tumors can realize the specific and efficient delivery of drugs in tumor cells, and are expected to become an effective drug delivery platform to overcome tumor multidrug resistance.

2.2 Redox-sensitive SRPDCs

Reduced glutathione (glutathione, GSH) is an important substance that determines the redox environment in organisms. The intracellular GSH concentration (about 2-10 mmol L-1) is about 100-1000 times that of the extracellular concentration (about 2-10 μmol L-1).

In addition, due to hypoxia, the concentration of GSH in tumor cells is at least 4 times higher than that in normal tissue cells, so the microenvironment of tumor cells is highly reducing.

Disulfide bond is a chemical bond that is sensitive to GSH in the environment. SRPDCs prepared by using disulfide bond as a connecting bond can exist stably in blood circulation. After entering tumor cells, under the action of higher concentration of GSH, the disulfide bond Responsive cleavage, releasing the active drug.

Hu et al. prepared disulfide-linked hydroxyethyl starch and DOX conjugates (HES-SS-DOX). The results of drug release experiments show that HES-SS-DOX can maintain stability (about 30% drug release in 24 h) in the simulated extracellular GSH level (about 2 μmol L-1), but when placed in simulated cells When the level of GSH (2 ~ 10 mmol·L-1) is in the environment, the drug release is accelerated (24 h drug release is about 85%).

The experimental results showed that compared with the drug solution, the distribution of HES-SS-DOX in tumor tissue was increased, the plasma half-life was prolonged, and it had better anti-tumor effect and less systemic toxicity.

2.3 Enzyme-sensitive SRPDCs

Compared with normal tissues, certain enzymes in tumor tissues, such as matrix metalloproteinase 2 (MMP2), cathepsin B, esterase, etc., are highly expressed to promote tumor growth, invasion or metastasis.

Abnormally expressed enzymes are not only an important basis for judging tumor types, but also serve as the material basis for the development of enzyme-triggered drug delivery systems.

Due to the strong specificity of enzymes for substrate selection and milder reaction conditions, the enzyme-sensitive and responsive controlled release system has the advantages of not easily causing drug leakage and destroying the drug structure during drug delivery.

Selecting highly expressed enzyme-degradable chemical bonds in tumor tissue to prepare PDCs can achieve tumor-targeted drug delivery and reduce drug distribution and toxicity in normal tissues.

2.3.1 MMP2-sensitive SRPDCs

MMP2 is an enzyme that is highly expressed in most tumor tissues and less expressed in normal tissues. The specificity of its distribution can be used in the development of tumor microenvironment-responsive drug delivery platforms.

Torchil-in’s research group chose paclitaxel (paclitaxel, PTX) as a model drug, PEG as a polymer material, and a polypeptide (PL-GLAG) that can be specifically degraded by MMP2 as a linker, prepared PEG2000-PLGLAG-PTX, and also synthesized Cell-penetrating peptide-PEG1000-phosphoaminoethanol (TATp-PEG1000-PE) is used to increase the ability of drugs to enter cells. After the mixed micelles prepared by the above two substances enter the tumor tissue, under the action of high-concentration MMP2, the tether can Responsive cleavage, PEG2000 is shed from the surface of the micelles, exposing the inner layer TATp, thereby assisting the efficient entry of drugs into tumor cells.

Compared with non-MMP2-sensitive micelles, the micelles showed good MMP2-dependent drug release behavior and better anti-tumor effect.

2.3.2 Cathepsin-sensitive SRPDCs

Cathepsin B (cathepsin-B) is an enzyme that plays an important role in tumor invasion and metastasis, and its expression in various tumors is 3-9 times that of normal tissues.

Therefore, selecting ca-thepsin-B-sensitive peptides as linkers to prepare SRPDCs can achieve tumor tissue-specific drug release.

Ducan’s group designed and synthesized a series of SRPDCs with ca-thepsin-B sensitive polypeptide (Gly-Phe-Leu-Gly) as the tether.

Among them, PEG-irinotecan (etirinotecan Pegol 326, NKTR-102) and Opaxio® (polyglutamic acid-paclitaxel, PGA-PTX) have entered phase III clinical research.

The results of drug release experiments showed that under the action of cathepsin-B, the release of the two drugs could reach 100%.

Clinical studies have shown that, compared with the drug solution group, NKTR-102 can significantly prolong the overall survival rate of breast cancer patients with brain metastases and reduce the inhibitory effect of irinotecan on neutrophils.

For Opaxi o®, studies have found that its efficacy is influenced by the patient’s estradiol levels, which are closely related to cathepsin B activity.

2.3.3 AZO-sensitive SRPDCs

The flora present in the human colon can secrete a variety of enzymes, one of which is azoreductase, which plays an important role in the colon.

In recent years, azoreductase-sensitive drug delivery carriers have been widely used in the development of colon-targeted preparations. Selecting chemical bonds that can be degraded by azoreductase as linkers can enable PDCs to have the performance of colon-targeted drug release.

Gao et al. linked camptothecin (CPT) through aromatic azo bonds to prepare HPMA-Azo-CPT. The results of in vitro degradation experiments show that HPMA-Azo-CPT can remain stable in the simulated upper digestive tract environment, and only in the cecal environment can there be significant and rapid drug release.

Pharmacokinetic experiments showed that, compared with free CPT, HPMA-Azo-CPT could increase drug distribution in the cecum and rectum.

Synthesized azoreductase-sensitive polyphosphazene-gemcitabine and polyphosphazene-methotrexate conjugates.

The results of in vitro drug release experiments showed that when the polymer was incubated with rat gastric contents, gastric mucosa and intestinal mucosa, the amount of drug released was small; when incubated with rat cecum contents, the drug release reached about 90% .

Therefore, this type of conjugate is expected to become an effective colon-targeted delivery platform, thereby improving the treatment efficiency of colon tumors.

2.4 Light-sensitive SRPDCs

In addition to the endogenous factors of the tumor environment discussed above, some exogenous stimuli, such as temperature, light, magnetic field, and ultrasound, can also be used for the development of stimuli-responsive nanocarriers.

Due to the particularity of drug loading methods, covalent bonds generally have poor responsiveness to temperature, magnetic field, and ultrasound. Therefore, most of the current research on exogenous stimulus-sensitive SRPDCs is based on the light-sensitive drug release mechanism.

At present, the light used in photosensitive therapy research is mainly near-infrared light, which has good penetration and safety.

Most of the light-sensitive SRPDCs contain photosensitizers and chemotherapeutic drugs that can generate reactive oxygen species (reactive oxygen species, ROS). Therefore, light-sensitive SRPDCs have the advantage of integrating the functions of photodynamic therapy and chemotherapy.

Liu et al. linked DOX to the polymer carrier-dextran (Dex-tran, Dex) through the ROS-sensitive thioketal bond, and the photosensitizer triphenylporphyrin (Por) was connected to the dextran molecule through the ester bond to prepare The light-sensitive Por-SA-Dex-TK-DOX that triggers the release of ROS reactive oxygen species was tested. Experiments showed that the release behavior of the drug from the conjugate and its growth inhibitory effect on tumor cells were light-dependent.

Thapa et al. connected the photosensitive material phthalocyanine to PTX through a photosensitive bond-ethylpiperazine bond, and prepared a folate (folate, FA)-targeted photosensitive FA-PEGn-Pc-L-PTX. Under light, the conjugate can effectively inhibited the growth of tumor cell SKOV-3, while no obvious cytotoxicity was observed under dark conditions.

2.5 Multiple stimulus-sensitive SRPDCs

Using multiple specific factors in the tumor environment to design multiple stimulus-sensitive SRP-DCs can more efficiently and precisely regulate the speed and location of drug release, thereby achieving precise targeted delivery of anti-tumor drugs.

Wei et al. synthesized branched bHPMA that could be degraded by cathepsin-B and prepared bHPMA-DOX with hydrazone bond-linked DOX.

The results of in vitro degradation experiments showed that the particle size of bHPMA-DOX decreased from 102 nm to about 8.6 nm under the treatment of 2. 4 μmol L-1 papain (acting the same as cathepsin B) and pH 5. 4 PBS, The relative molecular mass of the degradation product is about 23 000.

Because polymers with a relative molecular weight higher than 50 000 cannot be filtered by the glomerulus, there are toxic side effects related to accumulation in the body during application. The bHPMA is degraded into low-molecular-weight HPMA under the action of enzymes, so it not only ensures the tumor-targeting effect of the carrier, but also eliminates the hidden danger of accumulation of high-molecular polymers.

In addition, the in vitro drug release of bHPMA-DOX was pH-responsive, that is, at pH 5. 4, the drug release reached 75% within 12 h; while at pH 7. 4, the drug release was less than 20%.

The results of in vivo experiments showed that, compared with drug solutions, bHPMA-DOX could increase the distribution of DOX in tumor tissues, and had better anti-tumor effect and less toxic side effects.

3. SRPDCs for protein or peptide anti-tumor drug delivery

Protein and polypeptide drugs are important drugs for the treatment of tumors. However, these drugs have the disadvantages of being easily degraded by proteases in the blood, fast in elimination speed, and poor in stability. Linking proteins, polypeptide drugs and polymers to prepare polymer-protein (polypeptide) conjugates can improve the pharmacokinetic properties of such drugs and reduce their immunogenicity.

Talelli et al. designed a reduction-sensitive PGA-lysozyme conjugate with lysozyme as a model protein, PGA as a polymer material, and a disulfide bond as a link. Well shielded; lysozyme was rapidly released from PDCs and restored its activity after treatment with GSH.

According to the characteristics of excessive generation of reactive oxygen species around the mitochondria of most tumor cells, Qiao et al. reported a ROS-sensitive polymer-peptide conjugates (PPCs), in which, with β-fold peptide (KLVFF) through sulfur The ketal-linked PEG and the mitochondria-targeting cytotoxic peptide KLAK were bonded to the backbone material—polyvinyl alcohol, respectively. After the PPCs entered tumor cells, overexpressed ROS around the mitochondria could trigger the responsive shedding of the PEG hydrophilic layer. , thus causing the break of the hydrophilic-lipophilic balance of the carrier material, and then triggering the morphological change of PPCs, making it change from the state of nanoparticles to the shape of β-folded nanofibers, and then efficiently enter the mitochondria under the targeting effect of KLAK.

The changes in the morphology of PPCs are beneficial for KLAK to fully act on mitochondria from multiple directions in space and kill tumor cells efficiently; in addition, studies have found that PPCs only undergo morphological changes in ROS-overexpressing tumor cells, while their morphological changes in a variety of normal cells Keeping the original shape ensures the specificity and targeting of its anti-tumor effect.

As the first study to use intracellular ROS to trigger the morphological transformation of carrier materials, PPCs provide new ideas for the delivery of anti-tumor proteins and polypeptide drugs and the innovative treatment of cancer.

4. SRPDCs for the delivery of antitumor photosensitizers

Photodynamic therapy (PDT) is a new method of treating tumor diseases with photosensitizing drugs. Compared with traditional chemotherapy, it has the advantages of low toxicity, wide application range and good patient tolerance.

Nanocarriers have unique advantages in improving the transport and distribution of photosensitizers in vivo, increasing the tissue penetration depth of excitation light sources, and alleviating tissue hypoxia.

However, generally nanocarriers encapsulate photosensitizers densely and close to each other in space through physical encapsulation methods, so they have a very strong photoquenching effect, which limits their application in photodynamic therapy.

By preparing a polymer-photosensitizer conjugate that is sensitive to the tumor environment, it can release the photosensitizer quickly and in time after entering the tumor tissue, which can effectively avoid the photoquenching effect and improve the specificity of treatment.

Staegemann et al. used hyperbranched PEG as the carrier material and porphyrin as the photosensitizer to prepare pH-sensitive and disulfide-linked redox-sensitive polymer-photosensitizer conjugates, respectively. , the photosensitizer is dispersed on the surface of the nanoconjugate, which avoids the quenching effect related to the spatial proximity of the photosensitizer.

The research results showed that the two conjugates could rapidly release the photosensitizer under acidic conditions and the treatment of dithiothreitol (DTT), and had the best anti-tumor effect and no obvious toxic effect was observed.

Therefore, the research on photosensitizer-based SRPDCs will help promote the development of a new generation of efficient and safe photodynamic therapy.

5. Summary and Outlook

SRPDCs can release drugs in response to tumor tissue-specific endogenous or external stimuli, achieving the purpose of fixed-point, timing, and quantitative regulation of drug release.

Although a large number of reports on SRPDCs have emerged in recent years, the research on this type of conjugates is still mostly in the laboratory research stage.

Opaxi o® was once speculated to be the first SRPDCs to be marketed. However, clinical trial studies found that compared with other chemotherapy regimens, no significant pharmacodynamic advantages were observed in patients with non-small cell lung cancer.

In addition, when the Human Medicines Product Committee of the European Medicines Agency reviewed the drug’s marketing application, it also focused on the issues of drug uniformity and drug release and distribution behavior in the body.

In view of the above reasons, 180 days after the application was submitted, its research and development company withdrew the drug’s marketing application.

Promoting the transformation of new drug technology achievements and enabling patients to benefit from the development of pharmaceutical technology in a timely manner is the direct driving force and ultimate goal of pharmaceutical scientific research.

Therefore, in the research work, while pursuing multiple functions, we should focus on the bottlenecks that limit the application of SRPDCs technology, such as:
① The preparation process is too complicated, and it is difficult to obtain products with uniform quality and good reproducibility, which is the main problem that restricts this type of technology from entering clinical application;
② There is still a lack of systematic and reliable research and evaluation methods to investigate and evaluate the pharmacokinetic process of SRPDCs after entering the human body and the detailed mechanism of drug efficacy;
③Heterogeneity of the internal environment of the human body should also be considered. For example, individual differences in patients, tumor types, and disease states will cause non-specific distribution or level fluctuations of pH, GSH, and enzymes in the body. It is necessary to pay attention to whether the above factors It can lead to off-target effects of SRPDCs.

A safe and effective treatment system originates from rational design, selecting polymer materials that are safe for the human body, linking bonds that meet the requirements for stability, and improving synthesis and preparation methods. Obtaining preparations with uniform properties and accessible clinical applications is the key to optimizing the research on SRPDCs .

With the rapid progress of related disciplines and new technologies in pharmacy, as well as the continuous improvement of its design and preparation methods, SRPDCs are expected to become a promising platform for anti-tumor drug delivery.

Polymer-drug conjugates with target specific peptide ligands can provide a solution for selective and targeted drug delivery.

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1. Chinese Journal of Pharmaceutical Sciences, Volume 54, Issue 22, November 2019