Abstract
Despite recent advances in cancer therapy, the development of a clear strategy for targeted anti-cancer therapeutics has remained a major challenge in research. Given the vast complexity of cancer cells, conventional therapies are often ineffective due to dose-limiting toxicity and drug resistance. In an effort to develop innovative approaches to chemotherapy, enzyme instructed self-assembly (EISA)6 has recently been introduced as a novel strategy to selectively target cancer cells by exploiting the integration of enzymatic transformation and supramolecular self-assembly. Using this approach, supramolecular self-assembly produces nanofibers or hydrogels that inhibit tumors by interrupting metabolic processes, therefore inducing apoptosis. By harnessing the selective catalytic activity of enzymes overexpressed in cancer cell lines, it is possible to control and direct self assembly at a specific location, where it can then elicit its desired effects. Three representative examples of substrates of EISA are outlined, demonstrating this concept as a viable therapeutic for cancer treatment.2,4,5 In each example, EISA shows a high degree of selectivity for cancer cells in vitro and the cytotoxic effects are shown to be proportional to enzyme concentration in the cancer cells. Utilization of EISA holds great potential and further exploration of this process may lead to new directions for developing novel strategies for targeted anticancer therapies.
Introduction
To date, cancer has remained a major challenge to public health around the world and has become the second leading cause of death in the United States, preceded only by heart disease. By the end of 2016, there will be an estimated 1,685,210 new cancer cases diagnosed and 595,690 subsequent cancer deaths in the United States alone1. Unfortunately, the development of further improved treatment options have continued to challenge research efforts due to the vast complexity and fast evolution of malignant cells. Although considerable efforts have been made to further advance anti-cancer treatments, most marketed drugs come at the price of toxic side effects to the patients because of the poor selectively for the tumor, which subsequently leads to limitations in dosing when administering the drug. In addition to that, many conventional strategies for therapies rely on single molecule inhibition mechanisms that often fail due the complexity of the tumor environment and emergence of mutations and redundant pathways, ultimately leading to drug resistance of the malignant cells. With that in mind, it is essential to identify areas to improve the efficacy of targeted therapeutics to effectively inhibit cancer cells.
The process of molecular self-assembly has gained a lot of recent interest in research as a potential mechanism for biotherapeutics. Molecular self-assembly is a ubiquitous process in nature that facilitates the formation of a large range of biological complexes and is most often governed by enzymatic reactions. The reliance of this process on the enzymatic transformation of a precursor inspired researchers to exploit molecular self-assembly for the design of optimized targeted therapeutics. By targeting the catalytic activity of a specific enzyme associated with a disease state, researchers could direct and control self-assembly, affording new directions for selective delivery of therapeutics. In an effort to apply this approach to develop optimized strategies for the selective inhibition of cancer cells, enzyme instructed molecular self-assembly, or EISA, was introduced.6 Enzyme instructed molecular self-assembly can provide selective inhibition by exploiting the integration of enzymatic transformation of precursor amphiphiles and subsequent molecular self-assembly. In this approach, self-assembly will yield nanofibers or hydrogels that have potential to inhibit tumors by interrupting vital metabolic exchanges, resulting in global consequences that ultimately lead to apoptosis. By targeting the selective catalytic activity of enzymes overexpressed in cancer cells exclusively, it is possible to control and direct self assembly at a specific location where it can elicit its desired effects on the cell. Enzyme targets will convert a precursor amphiphile into a hydrogelator, yielding a more hydrophobic entity that is free to self assemble to form hydrogels. Specific enzymes like phosphatases or MMPs; which break/hydrolyze a phosphoester bond or peptide bond respectively, will remove hydrophilic or electrostatic blocking groups from non-assembling precursors and generate building blocks at the site of catalysis. The utilization of supramolecular hydrogels as cytotoxic assemblies and drug delivery agents is attractive because of their excellent biocompatibility. Because supramolecular hydrogels are formed from self-assembling hydrogelators by non-covalent interactions, subtle changes to the chemical structure of the precursors can affect the self-assembly structure and process, affording its diversity and versatility. Utilization of EISA holds great potential, ultimately giving rise to new directions for developing novel strategies for targeted anticancer therapies.
To validate the concept of enzyme instructed self-assembly for selectively killing cancer cells, three representative examples of substrates of EISA are outlined to illustrate the concept as a viable therapeutic for cancer. In the first example, Pires et al. reports the use of an aromatic carbohydrate amphiphile for the successful inhibition of osteosarcoma cell line, SaOs2, through the extracellular self assembly in situ, by membrane bound alkaline phosphatase.4 Second, Tanaka et al. reports a similar approach that instead uses extracellular MMP-7 enzymes to instruct an intracellular self-assembly of a peptide lipid to initiate cancer cell death of HeLa cells.5 Lastly, Liu et al. exploits the process of EISA to develop a novel Cisplatin prodrug that demonstrates enhanced tumor selective accumulation and reduced systemic toxicity, in vivo, with 4T1 bearing mice models.2 In each example, the process of EISA shows a high degree of selectivity for cancer cells in vitro and the cytotoxic effects are shown to be proportional to enzyme concentration in the cancer cells.
Results and Discussion The utilization of EISA as an emerging approach for developing cancer therapy promises great potential. To date, there have been numerous accounts of research that seek to further explore this concept through the development of various precursor amphiphiles that yield the formation of nanofibers upon enzymatic transformation. Each study validates the efficacy of EISA and provides evidence of its successful inhibition of malignant cells selectively in vitro. In the following, three representative examples of various substrates for EISA are outlined:
a. Extracellular Self Assembly of Carbohydrate Amphiphile by Membrane bound Alkaline Phosphatase
Pires et al. recently presented the development of an aromatic carbohydrate amphiphile, N-(fluorenylmethoxycarbonyl)-glucosamine-6-phosphate, as a substrate of alkaline phosphatase for molecular self assembly.4 The designed amphiphile precursor presents an aromatic moiety to lend to its ability to self assemble and a sugar moiety as a carbohydrate. The incorporation of a carbohydrate group is attractive as their abundance of hydroxyl groups affords highly hydrated gels, improving gelation process. In addition, carbohydrates are highly biocompatible, as they are one of the most abundant biomolecules and a major building element of life.
In their approach, Pires et al. demonstrated that their carbohydrate peptide precursor can successfully be converted into a self assembling hydrogelator upon dephosphorylation by membrane bound alkaline phosphatase (ALP) of osteosarcoma cell line, SaOs2 in situ. Through their results, they proved the process to be cell specific, demonstrating higher degrees of cytotoxicity in cells overexpressing ALP like SaOs2 in comparison to that seen with chondrogenic ATDC5 cell line exhibiting lower levels of membrane bound ALP. Thus, suggesting this system can be used in an anti-osteosarcoma strategy with minimal impact on the surrounding healthy tissue.4
Alkaline phosphatases, a hydrolase enzyme responsible for dephosphorylation of many biomolecules, have attracted attention as an enzymatic target because of its high catalytic efficiency and the frequency of dephosphorylation reactions that occur in vivo. Alkaline phosphatase generally exists in two populations within biological systems, either bound to the membrane of the cell or soluble in the extracellular matrix. As its has been found to be over expressed in some osteosarcomas, alkaline phosphatases have become widely used as a diagnostic marker in different pathologies, making it a valid target for localized cancer therapies.
To first assess the ability of the precursor to become dephosphorylated and yield hydrogels, gelation was evaluated using three different concentrations of precursor 1 (10, 15, 20mM) and various concentrations of ALP (25, 50, 75 U) in PBS solution at pH 7.4, examined by TEM image. Concentrations of 1 were chosen to be >5mM, which is the critical gelation concentration of 2. Before ALP addition, a transparent solution was observed and subsequently treated with ALP to yield a transparent gel phase after 1-5 hours, depending on the initial concentration of precursor and ALP. Observations suggest that gelation time is faster in the presence of higher concentrations of each. Once gelation was demonstrated, the resulting hydrogel was further characterized. The mechanical properties of the hydrogel were evaluated by rheology after 24 hours time post ALP addition. This data revealed that higher ALP concentration yield stiffer gels. These results were consistent with that seen by fluorescence emission. As the concentration of precursor increases, the intensity of the fluorenyl peak at 320nm quenches as self-assembly occurs which is indicative of efficient stacking within nanofibers. Additionally, HPLC was used to asses the ability of ALP to dephosphorylate 1 over 24 hours and the results revealed that the conversion rate of precursor to hydrogelator is controlled by enzyme concentration.
Next, to investigate the ability of the precursor to self-assemble in situ, experiments were done in cell suspension and cell monolayer of SaOs2 cells in the presence of various concentrations of precursor (0-20mM). Macroscopic hydrogels were observed after approximately 4 days at each initial concentration of precursor. The resulting gels were collected, dehydrated, and analyzed by SEM, revealing nanofibrillar structures organized around nucleating clusters. Interestingly, different morphologies were observed in the absence and presence of SaOs2 cells. Subsequent quantification of precursor to hydrogelator conversion showed a consistent 7mM concentration of hydrogelator in the culture medium after 1 day, independent of initial added quantity of precursor. This observation was consistent with the stable ALP activity observed by ALP quantification. The data confirms that hydrogel formation is limited to the concentration of ALP produced by the cells.
After confirming the ability of ALP to catalyze the conversion of precursor to hydrogelator to form nanofibers networks, MTS cell proliferation assays were used to assess changes to the metabolic activity induced by the hydrogels. In this experiment, SaOs2 cells were assayed with chrondrogenic ATDC5 cells to confirm cell specificity for cancer cells. ATDC5 cells exhibit lower ALP activity compared to SaOs2 cells and are commonly used in combination with SaOS2 in vitro as models of neighboring tissues, bone and cartilage. It was found that the presence of the precursor shows minimal affects the metabolic activity of ATDC5 cells, however, drastic decreases in metabolic activity were observed in the SaOs2 cells. To test whether the effect observed was a direct consequence of higher ALP expression exhibited on the SaOs2 line, cells were assayed in the presence of Pierce phosphatase inhibitor showing complete recovery of the metabolic activity. Although there was clear evidence of a direct relationship between ALP activity and cytotoxicity of precursor 1, it is known that ALP can be expressed differently within biological systems, localizing at the membrane or extracellular matrix as soluble or insoluble populations. By quantifying both membrane bound and extracellular ALP in SaOs2 and ATDC5 cells cultures using a p-nitrophenol assay, it was found that membrane bound ALP has 15-20 times higher values in SaOs2 and extracellular ALP has only 1.5-2 times higher values for SaOs2 compared to those of ATDC5, indicating membrane bound ALP is mainly responsible for the cytotoxicity of the precursor. Additionally, they confirmed that the conversion of the precursor to the hydrogelator by membrane bound ALP of SaOs2 cells results in the formation of pericellular “cage like” nanonets around the surface of the cells by SEM imaging. Thus, it is concluded that the dephosphorylation of precursor 1 by membrane bound ALP is responsible for cytotoxicity of 1 toward SaOs2 cells.
The results presented by Pires et al. demonstrate the successful use of carbohydrate derivatives for the formation of “cage like” hydrogels in situ catalyzed by membrane bound ALP on SaOs2 osteosarcoma cells. The formation of the hydrogels around the surface of the osteosarcoma cell line showed ability to reduce metabolic processes between cells, which will cause critical stress that could lead to apoptosis of the cancer cells. The use of carbohydrate amphiphiles offers diversity to the design of amphiphile precursors and promises new possibilities for developing novel cancer therapeutics based on EISA.
b. Intracellular Self Assembly of a Peptide Lipid Hydrogelator by Extracellular Matrix Metalloproteinase-7
Tanaka et al. presents an innovative approach, establishing the use of extracellular enzymes to instruct the intracellular self-assembly of peptide lipids to initiate cancer cell death.5 Specifically, the designed peptide lipid precursor undergoes enzymatic transformation catalyzed by secreted MMP-7 to produce a hydrogelator analogue that is subsequently internalized by the cancer cells. Once inside the cell, the hydrogelator is free to self assemble to form a network of nanofibers that critically impair cellular function leading to the expiry of multiple lines of cancer cells.
The strategy that converts precursors to hydrogelators is not limited the phosphatases as seen previously. In this design, Tanaka et al. reports the use of matrix metalloproteinase-7 to instruct self-assembly, designing a peptide lipid that upon hydrolysis yields a hydrogelator.5 Matrix metalloproteinase-7, or MMP-7, is an enzyme involved in the hydrolysis and subsequent breakdown of extracellular matrix in both normal physiological and disease processes like cancer cell metastasis. For this reason, it is excessively produced and secreted by many cancer cells as the high expression can help facilitate invasion by degrading the extracellular matrix and connective tissue. Thus, the utilization of this enzyme as a target for localized cancer therapies is attractive.
In this design, the precursor peptide lipid yields a hydrophobic hydrogelator analogue that can then be internalized by the cells and induce intracellular self-assembly. Precursor, ER-C16, exhibits a long alkyl chain of 16 carbons linked to a short peptide sequence. The 16-carbon palmitoyl group affords an enhanced ability for self-assembly in solution by providing a larger degree of hydrophobic interactions. Following that, the Gly-Gly-Gly-His amino acid sequence allows sites for hydrogen bond donor/acceptor pairs. The Pro-Leu-Gly-Leu sequence is the MMP-7 cleavage site, necessary to confer the ability of the precursor to self assemble. Finally, the cationic peptides at the C terminus (Arg-Lys) are essential to prevent the formation of nanofibers prior to enzymatic hydrolysis. Cleavage of precursor ER-C16 by MMP-7 will yield hydrogelator entity, G-C16, and a short peptide fragment by-product.
To confirm the ability of the precursor to induce gelation, TEM images were taken to observe self-assembly of 0.2wt% precursor in solution with the addition of 2ug/mL MMP-7. Before the addition of the enzyme, micelle-like morphologies were observed of the precursor in solution. After the addition of the enzyme, hydrogel was formed after 2 hours, revealing the presence of bundled nanofibers. Subsequent treatment of the hydrogel with 8M urea chemical denaturant caused dissociation of the aggregates, indicating the significance of hydrogen bond and hydrophobic interactions among the molecules.
After confirming the ability to self-associate in solution, self-assembly was also assessed in the presence of human cancer HeLa cell secreted MMP-7. First confirming the secretion of MMP-7 by the HeLa cells, a MMP-7 assay kit was used to quantitate the amount of enzyme in the supernatant of the culture medium. Results confirmed the presence of 2.0ug/mL MMP-7 in the supernatant of the HeLa culture media. Subsequent treatment of 1mL of supernatant with 3.6wt% precursor revealed gelation after 2-day incubation time. MALDI-TOF/MS analysis of the resulting hydrogel showed the presence of hydrogelator G-C16, proving the ability of HeLa secreted MMP-7 to catalyze the hydrolysis of the precursor to form G-C16 whose molecular self-assembly triggered hydrogelation.
Next, to test the selectivity of the precursor for cancer cells, a live/dead cell assay was used to evaluate the cell inhibitory effect of both the precursor and the hydrogelator on human cancer HeLa cells and MvE human dermal microvascular endothelial cells. Using calcein AM and ethiduim homodimer, confocal laser scanning microscopy reveals the presence of living cells, dyed green by the intracellular enzymatic hydrolysis of calcein AM, and dead cells, dyed red by the intercalation of ethidium homodimer to DNA. The authors found that both 0.02wt% precursor and 0.02wt% hydrogelator induced cell death of HeLa cells. In contrast however, the MvE cells were alive in the presence of precursor and killed in the presence of the hydrogelator. The results reveal the cytotoxicity of the precursor exclusively for the HeLa cells, while remaining non-toxic to the MvE cells and the cytotoxicity of the resulting hydrogelator to both cells.
To further confirm the cytotoxicity of the precursor and hydrogelator, MTT cell viability assays were performed on HeLa cells. The quantitative evaluation of cell viability in the presence of precursor and hydrogelator showed significant decrease in the HeLa cells. The addition of precursor ER-C16 and hydrogelator G-C16 resulted in <5.3% and <24% viability of HeLa cells. The peptide fragment by-product of hydrolysis was tested as well and showed no cytotoxicity. In the presence of the precursor, the cell viability of the HeLa cells fell to 27% after 30 minute and <17% after 1.5 hours, indicating a rapid killing process. In contrast, the rate observed in the presence of the hydrogelator was much slower. After 4 hours, the cells viability was still more than 20%, while the cell viability of precursor treated cells was almost zero. However, these results are not surprising, as it is understood that the hydrogelator exhibits long fibril formation, which would prevent uptake by cells. Thus, the presence of long nanofibrils could potentially impede the uptake by cells.
Next testing the effects of concentration of precursor on cell viability, HeLa and MvE cells were incubated with various concentrations of precursor, ER-C16. At 0.005wt% precursor, little effect on cell viability was observed for both HeLa and MvE cells. Increasing the concentration to 0.01 wt%, cell viability of HeLa cells dropped to 35%, whereas MvE cells were not affected. At 0.025 wt%, approximately 94% of the HeLa cells killed. Finally, at the highest concentration measured of precursor, 0.05 wt%, most all HeLa cells were killed and more than 80% MvE cells were killed as well. To explain these results, it is known that normal cells also produce and secrete small amounts of MMP-7, so it is not surprising to see a degree of cytotoxicity at high concentrations of precursor. The results presented confirm that the concentration of precursor strongly affected the cell viability and a large difference observed between the HeLa and MvE cells.
The viability of various cell lines were examined next to prove to selectivity of the precursor for cancer cells across a range of cells. Exposure to the precursor exhibited varying degrees of cytotoxicity to all tested cancer cells, including A431, SKBR3, and MCF-7 cells, and little effect shown on the normal cell types. To confirm the assumption that the cytotoxicity exhibited by the cancer cells was correlated to the amount of MMP-7 secretion, the MMP-7 assay kit was used again to determine the concentration of the enzyme in the culture medium of each tested cell. Results showed that cancer cells secrete significantly more MMP-7 in comparison to the normal cells. Specifically, HeLa cells exhibit six times the amount observed by MvE cells. The results confirm the correlation between the cytotoxicity and concentration of enzyme secreted by the cell.
Finally, the authors sought to prove the internalization of the hydrogelator by the cells and validate the intracellular self-assembly. Using fluorescence microscopy, HeLa cells were incubated with both the hydrogelator and an analogue of the hydrogelator bearing a NBD fluorophore at the end of the alkyl chain. Due to the structural homology between the two entities, formation of nanofibers was allowed and fluorescent visualization revealed the self-assembly inside the cells, suggesting the uptake of hydrogelator by HeLa cells. Fluorescence recovery after photobleaching, or FRAP, was initiated to test the fluidity of the intracellular environment. A small area of the cell is photo-bleached, and fluorescence recovery is monitored. However, after 40 minutes, the fluorescence had not recovered, indicating a high viscosity within the cell. This high degree of viscosity implies the intracellular hydrogelation by the hydrogelator. Previous literature has shown a relation of cell death to high intracellular viscosity, thus the authors concluded that the intracellular hydrogelation indeed could have caused the cell death seen previously. Using the lysate of dead cancer cells, authors further confirmed the presence of nanofibers inside the cell. Dead cells were collected, sonicated, and resulting cell lysate was analyzed. The lysate formed a hydrogel and MALDI-TOF analysis revealed the presence of the hydrogelator. These results further support the hypothesis that the cell death was in fact induced by intracellular nanofiber formation.
Overall, the work presented by Tanaka et al. further validated the use of EISA for selectively killing cancer cells. The results indicated that the cytotoxicity of precursor ER-C16 is the result of MMP-7 catalyzed hydrolysis. The conclusions are confirmed by the high correlation between the viability observed by the various cell lines tested in the presence of the precursor and the concentration of secreted MMP-7 by each.5 Furthermore, the proven internalization of the hydrogelator by the cells suggests a mechanism of induced cell death conferred by the high viscosity in the intracellular environment upon self-assembly.
c. Self Assembly of a Platinum Prodrug by Membrane Bound Alkaline Phosphatase and Controlled Platinum Release by Intracellular Glutathione
Liu et al. designed a novel self assembled Platinum (Pt) prodrug (c,c,t-[Pt(NH3)2Cl2(OH)2]) that undergoes molecular self assembly in situ, in the presence of membrane bound ALP and subsequently performs controlled release of a Pt(II) drug, like Cisplatin, under the reductive conditions of tumor cells.1 In this approach, the authors seek to achieve efficient drug retention by taking advantage of the enzymatic activity of ALP in vivo and trigger the formation of self-assemblies at the site of the tumor. Upon self-assembly, prolonged and sustained Pt(II) drug release can be achieved in response to the redox environment of the cells. The intracellular and extracellular environment of mammalian cells exhibit significant differences in terms of redox. The intracellular cytosol is considered a reducing environment due to the higher levels of glutathione content in comparison to the extracellular matrix. Because abnormal levels of glutathione are often linked to cancer, exploiting the difference in content could provide an excellent target in terms of targeted therapeutics.
In their design, the authors seek to develop and optimized approach to deliver Pt drugs, like Cisplatin, to the site of the tumor.1 Cisplatin is one of the first members of the class of Pt containing anti-cancer agents. The drug reacts in the body and binds to the DNA, causing the DNA crosslink and subsequently inducing apoptosis. However, the clinical use of Cisplatin is limited by its systemic toxicity and poor bioavailability during drug administration. Therefore, developing a way to improve the localized distribution is highly attractive as it has potential to not only improve the bioavailability, but also enhance the efficacy, and reduce systemic toxicity. Various approaches have been taken to prolong the half-life of the drug and reduce its side effect, but unfortunately most have failed due to additional biosafety concerns and lower anti-cancer efficacy. However, most recently, the concept of self-assembled prodrugs has emerged as a novel approach to deliver the therapeutic agents. A simple covalent attachment of a parent drug like Cisplatin to a precursor could generate a prodrug with increased selectivity by exploitation of the presence of enzymes that are up-regulated in tumor cells. This approach would confer high drug loading and enhanced sustained delivery.
To validate this concept, Liu et al. designed Prodrug 1 with the goal of improving the efficacy of Pt drugs by reducing the toxic side effects without compromising anti-tumor activity.1 Prodrug 1 exhibits a napthyl group, two phenylalanines, one lysine, one phosphorylated tyrosine residue, and a Pt(IV) complex prepared from commercially available Cisplatin and reacted with the amino group on the lysine residue. Upon dephosphorylation, the prodrug will self assemble at the site of the tumor and the Pt(IV) can be released as Pt(II) in the presence of glutathione inside the cell. The aromatic residues confer the ability of the resulting hydrogelator to self assemble, and the phosphate group on tyrosine prevents the precursor from forming nanofibers without first having undergone dephosphorylation by ALP. The Pt(IV) complex exhibits great potential as a prodrug for Pt(II) due to its high kinetic inertness, low off target reactivity, and comparable antitumor effects to Cisplatin.
First, evaluating the ability of the prodrug to self-assemble, 2mg of prodrug 1 were dissolved in buffer at pH 7.4, making a clear solution with a final concentration of 1.0 wt%. The hydrophobic content of the resulting hydrogelator confers its ability to undergo self-assembly through aromatic-aromatic interactions and hydrogen bonding interactions to form the subsequent hydrogel. Using far UV CD spectroscopy, the secondary structure of the self-assembling entities was determined. The CD spectra indicates the presence of beta sheet like structure associated with the formation of nanofibers. The solution solely of precursor showed the absence of secondary structure, indicating that the presence of ALP is facilitating the conversion of precursor to nanofiber formation.
Next, the authors sought to determine the ability of the prodrug to perform a controlled Pt(II) release under the reductive conditions of the tumor environment. To do so, the hydrogel of the prodrug was added to PBS buffer, pH 7.4 containing various concentrations of glutathione. The presence of glutathione stimulated the release of Pt(II) through a reduction reaction eliminating the axial dihydroxy ligands. To quantify the amount of Pt(II) released, a fluorescence emission spectra of a dichlorofluorescein solution in the presence and absence of Pt(II) was obtained. With this assay, the resulting PBS buffer solution was collected, and released Pt(II) was further reduced to Pt(0) in the presence of triphenylphosphine. The Pt(0) forms a complex with triphenylphosphine (Pt(0)-PPh3) that will then turn on the fluorescence of dichlorofluorescein through the deprotection reaction of the allylic group on oxygen. In this way, fluorescence intensity can be monitored to determine the Pt(II) concentration released from the hydrogel. It was found that the rate of Pt(II) release was increased with the concentration of glutathione in PBS buffer. In the presence of 5μM GSH, release was very slow, showing only 20% of drug released after 24 hours. This concentration acts as a representation of the content of GSH in extracellular plasma, proving that the Pt(IV) drug will not be released prematurely in the extracellular environment of the cell. To contrast, in the presence of 5mM GSH, representative of the GSH content inside cancer cells, a steady drug release behavior was observed and almost 100% Pt(II) was released in 24 hour. This data confirms the use of this self assembling prodrug to perform a controlled release of Pt(II) drug according to reductive conditions of cytosolic environment of the cell.
The cytotoxicity of the prodrug was then confirmed by MTT cell proliferation assay on HeLa human cervical carcinoma cell line and 4T1 mouse breast cancer cell line. After 24 hours, the cytotoxicity was low, confirming the kinetic inertness of the prodrug. However, after 48 and 72 hours, drastic decrease in the cell viability across both cell types was observed. Results confirmed that the cytotoxicity of the prodrug is dependent on the concentration and incubation time. To further investigate the mechanism for the observed decrease of cell viability, confocal fluorescence microscopy was used to examine the possible occurrence of apoptosis in HeLa cells. Apoptosis is the most widely investigated process of cell death induced by anticancer drugs, making it a viable assumption for the plausible mechanism of cell death. During apoptosis, intracellular capase-3 is activated and is known to be an important indicator of the cells entry into an apoptotic pathway. With this in mind, real time imaging was conducted using a fluorescent caspase-3 activation probe to monitor the occurrence of apoptosis. HeLa cells that were pretreated with prodrug 1 showed strong fluorescence after incubation with the caspase probe, confirming caspase-3 activity. In contrast, little to no fluorescence was observed in the control experiment in which the HeLa cells with pretreated with both fluorescent probe and caspase-3 inhibitors. The images confirmed the occurrence of apoptosis induced by the prodrug.
Finally, the antitumor activity of the prodrug was examined in vivo. In a comparative efficacy study, 4T1 bearing mice were divided into three subsets and treated with either PBS buffer, free Cisplatin, or 1 by direct intra-tumoral injection every 48 hours for 12 days. Analysis of changes to relative tumor volume showed that mice treated with Cisplatin and prodrug exhibited signs of significant tumor reduction. While tumors did being to grow again in prodrug post inject, the growth was much slower as compared to the rate observed with PBS treated mice. The results suggest that prodrug 1 can exhibit similar antitumor effects as free Cisplatin in vivo. To test whether the prodrug could elicit similar antitumor effects at a lower degree of systemic toxicity, changes in body weight of mice were monitored. These results proved the ability of the prodrug to maintain a relatively stable body weight in mice, increasing only gradually post injection time. In parallel, the biodistribution of each Pt drug was investigated between the tumors and the major organs using ICP-MS. Biodistribution profiles found that mice treated with prodrug 1 exhibited a higher proportion of Pt drug at the site of the tumor and smaller proportions localized at the major organs tested. These results were consistent with the earlier data confirming the low systemic toxicity of the prodrug. The tumor and major organs were subsequently removed from the mice and stained for histological analysis. Tumor tissue of the PBS group of mice exhibited tightly packed and dense tumor cells, indicative of rapid tumor growth. Tumor tissue of the Cisplatin and Prodrug groups showed evidence of apoptosis including nuclear shrinkage and extensive fragmentation. The various organ tissue showed no noticeable changes with prodrug. However, mice treated with Cisplatin showed significant pathological changes in several organs. These results further confirm the low toxicity of prodrug 1, making it a suitable substrate for long-term drug administration.
In summary, the integration of the Pt(IV) complex with the short peptide provided a novel prodrug that successfully exhibited both molecular self assembly and efficient controlled drug release in the response to the reductive conditions of the tumor environment. Utilizing this strategy showed improved efficacy for enhancing Pt drug accumulation at the site of the tumor and reduced systemic toxicity compared to free Cisplatin. This work further validates the use of EISA, while also highlighting its application as a novel delivery strategy of anti-tumor agents.
Conclusion The concept of EISA, as demonstrated in the outlined examples, shows great potential to provide promising changes to the development of targeted anticancer drugs. Used either separately or in combination with current therapies, EISA has demonstrated the ability to successfully direct and control self-assembly at specific locations where the enzyme of interest is overexpressed. In each example, the formation of nanofibers and hydrogels showed ability to reduce metabolic activity in the cells and induce critical stresses that can ultimately lead to apoptosis. As the cytotoxicity elicited by the formation of hydrogels is proportional to the concentration of the enzyme target, the utilization of enzymes as a biomarker target affords a high degree of selectivity to cancer cells.
The selective inhibition of cancers cells afforded by EISA is very attractive in its approach compared to conventional methods. The formation of nanofibers can effectively inhibit the cancer cell on a global scale by efficient modulation of the cellular environment. For example, in the presence of pericellular self-assembly, nanofibers form around the surface of the cell, essentially entrapping the cell as a whole. This isolation disrupts the microenvironment of the cell by blocking critical intercellular communications and metastasis. Alternatively, intracellular nanofibers confer large sizes within the cell that have potential to block multiple cellular pathways and interrupt protein interaction networks. These large sizes also disfavor efflux, a pro-survival mechanism that could decrease the concentration of a conventional small molecule chemotherapy agent. In this way, the self-assembly of intracellular nanofibers can prevent cell survival mechanisms, which will ultimately lead to cell death. This process of inhibition is also highly biocompatible. Due to the supramolecular nature of the nanofibers, subsequent dissociation to innocuous monomers can easily be accomplished after inhibiting cancer cells, greatly reducing the systemic toxicity observed with conventional methods.
The use of EISA extends beyond the scope of cancer therapy and can be exploited as a biotherapeutics strategy for a number of conditions. Since this process takes place in physiological conditions, it application in biomedicine is not surprising. By simply conjugating therapeutic agents with a precursor amphiphile, hydrogelators can be developed to treat a variety of conditions. For example, EISA has found several applications in fields of molecular imaging, anti-bacterial, anti-inflammatory therapeutics, etc.
Although the development of EISA is still in its infancy, it is clear that the process holds great potential. Further exploration of EISA will optimize this process for the future development of anticancer therapies that consider the immense complexity of cancer. The utilization of EISA for selective inhibition of cancer cells ultimately provides an unprecedented direction in cancer research for the prospective development of novel biomimetic strategies to optimize conventional immunotherapies used to date.
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