Biomedical Application of Cyclodextrin Polymers Cross-Linked via Dianhydrides of Carboxylic Acids

Abstract

Cyclodextrin-based nanosponges (CD-NS) are a novel class of polymers cross-linked with a three-dimensional network and can be obtained from cyclodextrins (CD) and pyromellitic dianhydride. Their properties, such as their ability to form an inclusion complex with drugs, can be used in biomedical science, as nanosponges influence stability, toxicity, selectivity, and controlled release. Most pharmaceutical research use CD-NS for the delivery of drugs in cancer treatment. Application of molecular targeting techniques result in increased selectivity of CD-NS; for example, the addition of disulfide bridges to the polymer structure makes the nanosponge sensitive to the presence of glutathione, as it can reduce such disulfide bonds to thiol moieties. Other delivery applications include dermal transport of pain killers or photosensitizers and delivery of oxygen to heart cells. This gives rise to the opportunity to transition to medical scaffolds, but more, in modern times, to create an ultrasensitive biosensor, which employs the techniques of surface-modified nanoparticles and molecularly imprinted polymers (MIP). The following review focuses on the biomedical research of cyclodextrin polymers cross-linked via dianhydrides of carboxylic acids.

1. Introduction

Cyclodextrins (CDs) are conical, truncated macrocycles; the α-, β- and γ-CD consist of six, seven, and eight α-D-glucose units, respectively. All α-D-glucopyranose molecules in the ring are connected by α-1,4-glycosidic bonds [1,2]. CDs can be transformed into cyclodextrins polymers, which are characterized by high molecular weight. Most of them are insoluble in water, but some of the polymers are water soluble [3,4,5,6,7,8,9]. The solubility of cyclodextrin polymers mainly depends on the molecular weight. It is also affected by the nature and type of functional groups found in linkers used in this kind of polymers. Currently, there are four basic polymer groups: poly(pseudo)rotaxanes, grafted polymers, linear and cross-linked polymers, each of which has a different topology [10,11,12,13].

Cyclodextrin-based nanosponges (CD-NS) are cyclodextrin polymers with a high degree of cross-linking and a three-dimensional network. They are insoluble, swelling materials with complexing properties, and nanometric porosity. The swelling ability of cyclodextrin-based nanosponges depends on the degree of cross-linking: when the density of cross-linking is lower, higher water uptake is observed [14,15]. A multi-functional monomer, cyclodextrin, needs to be treated with a dual or multi-functional cross-linking agent to obtain this category of polymers. This results in the formation of a three-dimensional nanoporous structure with a network of covalent bonds [14]. The cross-linking agents may include diisocyanates, dianhydrides, diglycidyl ethers, or compounds with an active carbonyl group.

CD-NS can form typical CD inclusion complexes, as in the case of CDs themselves, as well as non-inclusion complexes. This leads to the creation of larger spaces in the polymer structure. In these spaces, the polymer can bind molecules larger than the cavity of the free CD. This phenomenon is possible due to the presence of lipophilic cavities of CD monomers and hydrophilic channels of the porous structure of CD-NS [15,16].

The word “nanosponge” was first used by DeQuan Li and Min Ma in 1998 when they cross-linked β-cyclodextrin using diisocyanates [17]. The resulting network was insoluble and showed a very high adsorption efficiency for several organic impurities. For example, p-chlorophenol has almost been completely removed from wastewater even at the parts per billion level (ppb) [15]. Eventually, Trotta (2011) introduced synthesis methods and other applications of CD-NS, among others, in pharmacy (controlled release of drugs), catalysis, cosmetics, agrochemistry, etc. [18,19,20]

2. Methods of Synthesis of CD-NS

There following are the four main techniques to prepare CD-NS [16]:

2.1. Melting

This method focuses on treating CD with a cross-linking agent until melting. All the ingredients are homogenized and then heated at 100 °C. Then, everything is mixed at maximum speed on a magnetic stirrer for five hours and, after this process, the mixture is left to cool. Washing is repeated several times to remove unreacted substrates and byproducts [21].

2.2. Solvent Method

This method involves the use of a solvent, such as dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), to dissolve the cross-linking agent. In the contrast to melting, cyclodextrin is treated with a polar aprotic solvent, and this mixture is added in excess to the solution of the cross-linking agent. This method uses different temperatures from 10 °C on the reflux of the solvent for achieving a variable time range. The final product is obtained by the addition of a dispersion solution to distilled water [22].

2.3. Ultrasound Synthesis

Here, the CD is mixed with a cross-linking agent in a flask placed inside the ultrasonic bath filled with water. The bath parameters have been set to water temperature: 90 °C; sonication: for five hours. The process is carried out without the solvent [23].

2.4. Microwave Synthesis

Microwaves can reduce the reaction time of CD with the cross-linking agent. The obtained NS are characterized by a high degree of crystallization. Compared to ordinary heating, the reaction time for this process is reduced four times. In addition, it enables the distribution of one-sized particles with homogenous crystallinity [24].

3. Generation of CD-NS

CD-NS can be divided into four generations in regards to their properties [14,25,26].

First-generation. NS are obtained by a simple reaction of cyclodextrin with a cross-linking agent. They can be grouped into four types depending on the functional group of a given cross-linking agent [14]:

• Urethane (or carbamate) CD-NS—are obtained from CDs cross-linked via diisocyanates and are characterized by a rigid structure, high degree of resistance to chemical degradation, and small range of swelling in aqueous and organic environments. For example, a cross-linking agent is hexamethylene diisocyanate or 2,4-diisocyanate toluene [17];
• Carbonate CD-NS—are formed from CDs cross-linked via active carbonyl compounds; for example, 1,1′-carbonyldiimidazole, triphosgene, and diphenyl carbonate. These nanosponges are characterized by short cross-linking bridges, limited swelling capacity, and good stability in the acidic and weak in the alkaline environment [27];
• Ester CD-NS—are synthesized from CDs and dianhydrides or di/polycarboxylic acids. The examples regarding, pyromellitic dianhydride (PMDA), ethylenediamine-tetraacetic dianhydride (EDTA), butane tetracarboxylic acid dianhydride, and citric acid are used. Unlike the previous two types, the ester CD-NS can absorb significant amounts of water and form hydrogels. The degree of cross-linking affects the swelling capacity due to water absorption. A smaller degree of cross-linking can lead to the absorption of more water. In addition, less chemically stable as it can be more easily hydrolyzed in an aqueous environment than urethanes or carbonates [28];
• Ether CD-NS—are the products of the reaction between cyclodextrins and cross-linking agents with epoxide groups. Examples of such compounds regard epichlorohydrin, bisphenol A diglycidyl ether, ethylene glycol, and diglycidyl ether. Even though epichlorohydrin is toxic, most research still focuses on it. In this case, CD-NS exhibit high chemical resistance and controlled swelling capacity [29,30]. Their synthesis is carried out in an alkaline environment [31].

Second generation. Polymers with new properties and applications can be obtained by introducing different moieties into the CD-NS structure. Functional groups can be introduced in the following three ways: functionalization after cross-linking, functionalization before cross-linking, and functionalization simultaneously with cross-linking. In the first case, mainly the polymer surface is modified whereas, in other situations, the entire NS molecule is functionalized. However, the introduction of side groups into cyclodextrin molecules, prior to polymerization, can decrease the degree of cross-linking or even prevent it [14,32].

Third generation. Stimuli-sensitive nanosponges respond to external changes by changing their properties; e.g., shape, color, and permeability. This ability is derived from the behavior of the stimulus at the molecular, supramolecular level, or even by its morphology. NS can be stimulated by the temperature, electromagnetic field, redox potential, and pH. By controlling the relevant parameters associated with stimuli, it can lead to the controlled transport of substances or release of electric charge. For example, CD-NS can behave as hidden carriers and release the drug under appropriate physiological conditions, thus extending the lifetime of the drug [14,33].

Fourth generation. Molecularly imprinted polymers (MIP) are based on CD resulted from the non-covalent interactions between the guest molecule (template) and the monomer in the presence of a cross-linking agent during polymerization. The template gives nanosponges the ability to recognize molecules. Later, the template is removed, and the remaining voids adjust to the shape and size of the target molecule. The obtained MIP has specific molecular recognition, high selectivity, and affinity to the target molecule [14,20,34].

4. Biomedical Applications

Recently, more and more researchers have focused on the cyclodextrin polymers cross-linked via carboxylic acids dianhydrides, mainly in the biomedical aspects [18,19,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38]. The most common cross-linking agent for these purposes is PMDA [14,35]. In addition, dianhydride EDTA is used in the protection of the environment [28].

4.1. Drug Delivery

Cyclodextrins can be administrated by oral, nasal, dermal, ocular, and intravenous drug delivery systems. Lipophilic drugs are delivered in the aqueous medium via CD to the site of the drug action. When the lipophilic barrier is overcome, the drug is transported in water followed by diffusion of the inclusion complex (disintegration) and drug permeation through the biomembrane [36].

4.2. Simple Oral Complex Drug-NS-PMDA

Meloxicam (Figure 1) is a painkiller and anti-inflammatory drug. Shende et al. decided to encapsulate this drug in NS cross-linked via PMDA, due to its limited solubility in water and the instability. To determine bioavailability, they studied the degree and the rate of solubility and drug stability in vitro and in vivo [37]. In addition, analgesic and anti-inflammatory activities were tested. Drug release tests with NS demonstrated controlled release of the substance for 24 h. The solubility of the drug increased significantly, and its rate decreased with less crystallinity degree of meloxicam. The complex was stored for three months, and subsequent characterization confirmed the absence of any significant changes in the drug release, particle size, and the efficiency of encapsulation. The last tests have indicated the increase in the analgesic and anti-inflammatory activities of the meloxicam-NS system in relation to the drug itself. Therefore, this type of polymer is considered a promising and interesting meloxicam carrier [37].

Later, in 2016, Shende’s group invented and tested nanosuspensions formed from the complexation of lansoprazole (Figure 2), a proton pump inhibitor used in the treatment of gastric ulcer, gastroesophageal reflux disease, and Zollinger-Ellison syndrome. The group carried out tests on solubility, chemical stability, and in vitro drug release to assess the effect of the NS. In comparison to the pure drug and the complex drug-NS, a significant improvement of solubility of over 40% was noted. Increased stability resulted in increased protection against acid degradation in the stomach. Additionally, in vitro studies have demonstrated the controlled release of lansoprazole, which is much more satisfactory than the release of pure drug. Lansoprazole in NS can be considered as a promising method in the treatment of gastric ulcers [38].

Rosuvastatin (Figure 3) prevents high-risk cardiovascular disease by inhibiting cholesterol biosynthesis. The basic limitation leads to low bioavailability of about 20% because of the poor solubility of the drug. Gabra’s team, in 2018 complexed rosuvastatin in CD-NS by lyophilization and then tested the in vitro drug release, pharmacokinetics, and bioavailability. The same tests were carried out with a commercial rosuvastatin, rosuvastatin suspension, and the results were compared. Pharmacokinetic studies of the complex have indicated the increased absorption and higher concentration of the drug over a long period of time compared to the other tests (rosuvastatin without a complex). Thus, the data showed an increase in the oral bioavailability of the drug NS complex, which is a promising alternative to the commercial drug [39].

In the case of HIV treatment by rilpivirine (Figure 4), basic problems include poor solubility and dissolution rate that results in low bioavailability (32%) [40]. Due to this fact, research on the improvement of bioavailability via PMDA-NS was undertaken by Rao et al., 2018. In addition, the ternary complex was formed with drug-NS by adding tocopherol polyethylene glycol succinate. Based on the results of in vitro solubility and in vivo pharmacokinetics in rats, a two-fold increase in the bioavailability for binary and ternary complexes was discovered [40].

Similar studies were carried out on the PMDA-NS complex with curcumin (Figure 5) by Pusphaltha and colleagues in 2018. The drug has a potential anticancer effect, but also low solubility and photostability. The effect of complexing was studied carefully. Changes in the solubility, the release rate of the active substance, photodegradation, and cytotoxicity were analyzed. The first test indicated a significant improvement in water solubility. The second test showed a 16-fold increase of in vitro release in comparison to the pure curcumin. Besides this, the photostability of curcumin was also found to have improved significantly by up to 1.7 times. In addition, increased in vitro cytotoxicity was confirmed for MCF-7 (breast cancer cell line). Based on the results, nanosponges were found to be an effective curcumin nanocarrier [30].

Pushpalatha and other researchers have studied the effects of resveratrol (Figure 6) in the same year. Resveratrol is considered a potential anticancer drug, unfortunately, it has poor solubility, low bioavailability, and is chemically unstable due to its photosensitivity. Thus, the drug was encapsulated to the improvement of solubility, reduction in photodegradation, and accelerated drug release. Bioavailability was increased almost twice the time in comparison to the pure resveratrol at the same dose. In addition, increased cytotoxicity on the MCF-7 cell line was determined, consequently, the IC₅₀ value significantly decreased. Thus, carboxylate cross-linked β-cyclodextrin cross-linked via pyromellitic dianhydride can be considered as an effective vehicle in the oral drug delivery system of resveratrol [29].

The first in vivo tests of acute and repeated dose toxicity of selected β-CD-NS were carried out by Shende et al. in 2015. The obtained polymers were administered orally to rats for 28 days. Studies have shown no significant hematological and biochemical changes. Additionally, histopathological examination indicated the lack of damage to the most important organs: liver, kidneys, stomach, and intestines. Based on the results, it can be said that CD-NS are safe at selected doses for rats and show good biocompatibility. These nanosponges can be considered as a modern drug vehicle for improving the therapeutic effect; however, more extensive research is required [41].

5. Molecular Drug Targeting

The first studies on PMDA-NS for molecular targeting of cancer were conducted by Trotta and others in 2016. The team obtained a stimuli-sensitive NS in a simple reaction of β-cyclodextrin with pyromellitic dianhydride and 2-hydroxyethyl disulfide. When the disulfide bridge reacts with the thiol group of glutathione (GSH) (Figure 7), the concentration is higher in chemoresistant tumor cells than in the healthy cells. To determine the capacity of GSH-NS and the response to GSH at intracellular concentrations, doxorubicin was selected as the model anticancer drug. The obtained polymer can absorb water and shows a tendency to be cleaved in an aqueous solution of GSH due to the reducing agent. The in vitro drug release was very slow and prolonged over time. In addition, doxorubicin release was found to be proportional to the glutathione concentration, and a greater number of disulfide bridges increased the proportional release of the drug. Then, the biological tests were carried out on HCT-15 (colon tumor cell line), HepG-2 (hepatocyte tumor cell line), and A2780 (ovarian tumor cell line). Based on the results, the increased inhibition was confirmed for GSH-NS. The use of NS tested may be an interesting strategy in molecular targeted therapy. In this way, the in vivo side effects and the therapeutic dose can be reduced [42].

Later, Daga et al. in 2016, developed studies on the complex of doxorubicin (Figure 8) with GSH-NS. In vitro tests on cytotoxicity, DNA damage, and in vivo biodistribution, pharmacokinetics, and growth of cancer cells of rats were performed. The first test showed increased toxicity of the drug, which was related to cells with a high concentration of glutathione. Microscopic and cytofluorometric studies showed an increased penetration of the glutathione sensitive polymer in relation to a pure chemotherapeutic one. The exposure of the cells indicated increased effectiveness of the drug at low doses. DU145 (prostate cancer cells) and HCT-116 (colon cancer cells) showed increased efficiency for the drug NS; however, PC-3 (prostate cancer cells) and HT-29 (colon cancer cells) showed no significant differences for the pure drug or the complex. Increased DNA damage in cells with high levels of GSH has been confirmed for the complexed drug by the comet assay [43]. It is assumed the increase in cell deaths is caused due to the formation of DNA adduct as well as topoisomerase II poisoning. In vivo tests indicated an accumulation of pure doxorubicin and complex in the liver, spleen and, heart; little was found in the lungs and the prostate. Additionally, tumor growth was found to be limited and the proliferation of positive cells reduced for GSH-NS. Based on the results, it can be said that GSH-NS enhanced the chemotherapeutic effect of doxorubicin in cells with high glutathione concentration and no significant toxicity to organs. This type of drug delivery seems to be effective in targeting chemoresistant tumors. The effective dose of the drug and its systemic side effects were found to be reduced [18].

In 2018, Momin’s team decided to study further GHS-NS and its effect on erlotinib (Figure 9). The drug is used to treat lung cancer but its oral bioavailability is limited due to the poor solubility, instability in the gastrointestinal environment, and extensive first-pass metabolism. Therefore, the complexed drug and standard formulation were examined to determine the effect of GSH-NS. The tests were carried out for stability, in vitro drug release, in vitro, cytotoxicity on A549 (human lung carcinoma cells), pharmacokinetics, and biodistribution in mice (in vivo). The stability of the complexed drug was increased, and the release was found to be slow and prolonged over time. The concentration of the released drug was found to be proportional to the concentration of glutathione. Nanosponges with erlotinib exhibited improved cytotoxicity and inhibition of tumor growth by 97.5% compared to the pure drug with an inhibition of 48%. This indicates the encapsulated erlotinib manifested the nature of molecular targeting to the tumor site. The highest concentration of the drug was found in the lungs; it was negligible in other organs, such as the heart, kidneys, liver, or spleen, and lacked completely in the brain. Thus, a reduction of the side effects on non-targeted cells and reduction of the therapeutic dose were observed [19].

Strigolactones are derivatives of carotenoids, plant hormones with anticancer properties. Two of these analogs, namely MEB55 and ST362 (Figure 10), inhibit the proliferation of prostate cancer cells. The basic problem is poor solubility in water and stability at physiological pH. Therefore, Argenziano and others in 2018 studied these strigolactones that were complexed with GSH-NS. The complexation protected against chemical degradation and achieve a six-fold increase in solubility in water. Studies on the kinetics of drug release from NS showed pH to lie in the range of 5.5 to 7.4 and that the presence of glutathione accelerates the release of the active substance. Based on the evaluation of NS and pure drug effects on DU145 and PC-3, the complex increased inhibition of proliferation only for DU145. Thus, it can be said that GSH-NS is a useful tool in the targeted treatment of prostate cancer and improves the therapeutic effect of strigolactones [44].

6. Dermal Transport

PMDA-NS was first used as a multifunctional semisolid ingredient for drug delivery to the skin by Conte and the others in 2014. The effect of NS on the solubility and photostability of the photosensitizer benzoporphyrin-derivative monoacid ring A (BPDMA) and all-trans retinoic acid (ATRA), as well as the effect of diclofenac on skin permeation, were investigated [45].

BPDMA (Figure 11) is used as a photosensitizer in the photodynamic therapy (PDT) of age-related macular degeneration. However, its application is limited due to the very low solubility in water, resulting in its ineffective aggregation state. Moreover, it shows poor solubility, low-light absorption, and low singlet oxygen quantum yield. Based on the obtained data, it was found that complexation of PMDA-NS prevented aggregation in the aqueous solution, reduced photobleaching during irradiation, and did not have any effect on the generation of singlet oxygen [45].

In the second case, the effect of ATRA stabilization in gel formation was investigated by the polymer. During irradiation, the ATRA degraded to 13-cis and 9-cis retinoic acids (Figure 12). The use of CD-NS increased the half-life approximately by 200-fold compared to ATRA in ethanol. Therefore, NS can be applied as an excipient in water-based formulations to stabilize photosensitive molecules [45].

Diclofenac (Figure 13) belongs to the group of non-steroidal drugs with anti-inflammatory properties. This complex NS drug was investigated in local treatment. Typically, diclofenac is formulated as an ointment, gel, or cream-gel because its composition affects the ability to penetrate the skin. Conte et al. found, that CD-NS does not significantly affect diclofenac skin penetration especially in aqueous solutions. However, it is believed that the administration of the drug can be controlled in a semisolid preparation by adding PMDA-NS [45].

In conclusion, gels based on NS cross-linked via pyromellitic dianhydride have the potential for local drug delivery because of their ability to increase the solubility of lipophilic drugs in semisolid formulations containing water, improve photostability, and modulate their transport. In addition, a significant decrease in the permeating dose and increase in the location of the drug in the stratum corneum and viable epidermis was detected, indicating NS are promising multifunctional ingredients in mono and biphasic preparation for local applications [45].

7. Oxygen Therapy

In recent years, the frequency of heart failure (HF) has increased all over the world. HF appears due to myocardial infarction (MI) followed by ischemia and reperfusion. Therefore, in 2018 Femminò and others used PMDA-NS to improve controlled oxygen transport in the treatment of cardiovascular diseases [46].

Nanosponges were saturated with pure gaseous oxygen. Red blood cells were tested for hemolytic activity and only small hemolysis was observed, indicating the biocompatibility of the formulation. Slow-release of stored oxygen was observed with prolonged and constant release kinetics. During the stability studies, in the ischemic buffer and special medium of the H9c2 (a cell line of cardiomyoblasts), no changes were detected in oxygen concentration. Additionally, no aggregation was expected due to insignificant changes in electrokinetic potential. Subsequently, the H9c2 cell line was treated with nanosponges containing oxygen or nitrogen with various concentrations under normoxic conditions (20% oxygen). During the nitrogen studies, no changes were detected in cell viability regardless of the gas concentration. However, the use of oxygen resulted in a significant increase in lifespan, regardless of its concentration. The next assays determined the effect of NS administration before and after application H/R (hypoxia (5% CO₂ and 95% N₂)) followed by reoxygenation. Regardless of the NS-oxygen dose given prior to the test, damage by H/R was limited. However, administration after application of H/R indicated that the cells showed limited viability. On the other hand, NS-N₂ given before and after H/R did not fulfill the protective function [46].

Cross-lined polymers were found to be a key factor in the storage and release of oxygen. The use of nanotechnology is beneficial for the treatment of acute myocardial infarction as NS-oxygen protects cardiomyoblast cells in the normoxic as well as H/R condition. In addition, the “top-down” method in the storage of oxygen in NS, e.g., high-pressure homogenization, can be applied to reduce the nanosponge’s size. Reduced CD-NS can form an aqueous nanosuspension with potential use in intravenous administration. In conclusion, this solution can be used to limit the damage caused during surgery or reperfusion treatment [46].

8. Potential Scaffolds

In 2018, Cecone and colleagues applied electrospinning on water-soluble PMDA-NS to give insoluble fibrous mats. Additionally, the obtained fibers were found to be thermally cross-linked. Solubility assays were carried out: the first in aqueous solution and the second in phosphate buffer at room temperature. In both cases, the examined mats turned out to be insoluble and retained their morphology. Based on the data, the obtained research object can be said to have potential in regenerative medicine as biocompatible scaffolds after appropriate assays [47].

9. Biosensors

Antigliadin antibody is used as a diagnostic tool to monitor celiac patients’ compliance with gluten free-diet. Wajs and others in 2014 synthesized NS as a signal enhancement tool in colorimetric and electrochemical assays related to the enzyme. The surface of the PMDA-NS molecule was modified by a covalent bond between antibodies and the carboxylic acid group. Then, this polymer encapsulated the particles of horseradish peroxidase. The complex was examined for its use in the aforementioned assays, and the Sandwich format was used for evaluation. Gliadin as an antigen was immobilized, and anti-gliadin antibodies labeled with horseradish peroxidase as well as the complex of NS were captured. Studies have shown that nanobioconjugates of this type may be used to develop ultrasensitive biosensors [48].

Deshmukh et al. in 2015 conducted a biomimetic estimation of glucose level by non-molecular imprinted polymer (NIP) and molecular imprinted polymer (MIP) PMDA-NS. D-glucose-6-phosphate was used as a template molecule for imprinting. In the beginning, NIP and MIP were characterized and their ability to bind glucose in buffer was compared. It was found that MIP has a much better affinity for glucose than NIP. Therefore, MIP was used for the next studies on the estimation of the blood glucose level in animals. The test object was rats that were divided into three groups based on the obtained drink: control (distilled water), aqueous glucose solution, and MIP (then glucose). All groups were subjected to blood tests. It turned out that in the third group, there were no changes in the glucose concentration (due to the ability of MIP to bind glucose). In addition, MIP were found to have greater affinity than NIP because diffusion of the template in the formed cavity occurs due to its large surface [49].

10. Conclusions

CD-NS cross-linked via PMDA are a new type of polymers whose application can be found in biomedicine. Their main advantages are biocompatibility, improvement of lipophilic compound solubility, increased stability, controlled release of the active substance, and reduction of toxicity. These factors are the reason for continuous research on these nanosponges. Their best area of application seems to be a pharmacy in oral drug delivery, especially molecular targeting of cancer. On the other hand, there may exist other possible ways to deliver active substance; e.g., dermal transport. Additionally, surgical medicine can be a tool for cardiac surgery to prevent heart failure or biocompatible scaffolds in regenerative medicine. Lastly and interestingly, they can be used as diagnostic tools for celiac disease or glucose level assays that rely on supramolecular phenomena. To sum up, CD-NS cross-linked via PMDA are novel tools for biomedical science but require more research in vivo and further clinical trials. Despite this, there are still undiscovered applications that can be used to revolutionize life sciences.