Chaperonin-GroEL as a smart hydrophobic drug delivery and tumor

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Chaperonin-GroEL as a smart hydrophobic drug delivery and tumor targeting molecular machine for tumor therapy Yi Yuan, Chong Du, Cuiji Sun, Jin Zhu, Shan Wu, Yinlong Zhang, Tianjiao Ji, Jianlin Lei, Yinmo Yang, Ning Gao, and Guangjun Nie Nano Lett., Just Accepted Manuscript • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Chaperonin-GroEL as a smart hydrophobic drug delivery and tumor targeting molecular machine for tumor therapy Yi Yuan1‡, Chong Du1,2‡, Cuiji Sun1‡, Jin Zhu1, Shan Wu3, Yinlong Zhang1,4, Tianjiao Ji1, Jianlin Lei3, Yinmo Yang2, Ning Gao3,5* and Guangjun Nie1* 1

CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology, Beiyitiao 11, Zhongguancun, Beijing 100190, China 2

Department of General Surgery, Peking University First Hospital, Beijing 100034, China

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Beijing Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

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College of Pharmaceutical Science, Jilin University, Changchun 130021, China

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State Key Laboratory of Membrane Biology, Peking-Tsinghua Joint Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China ‡These authors contributed equally to this work. *Correspondence to: [email protected] or [email protected] Abstract: The targeted delivery of hydrophobic therapeutic drugs to tumors is one of the major challenges in drug development. The use of natural proteins as drug delivery vehicles holds great promise due to various functionalities of proteins. In the current study, we exploited a natural protein, GroEL, which possesses a double layer cage structure, as a hydrophobic drug container, which is switchable by ATP binding to a hydrophilic status, to design a novel and intelligent hydrophobic drug delivery molecular machine with a controlled drug release profile. When loaded with the hydrophobic antitumor drug, Doxorubicin (Dox), GroEL was able to shield the drug from the aqueous phase of blood, releasing the drug once in the presence of a critical concentration of ATP at the tumor site. Unexpectedly, we found that GroEL has a specific affinity for the cell structural protein, plectin, which is expressed at abnormally elevated levels on the membranes of tumor cells, but not in normal cells. This finding, in combination with the ATP sensitivity, makes GroEL a superior natural tumor targeting nanocarrier. Our data show that GroEL-Dox is able to effectively, and highly selectively, deliver the hydrophobic drug to fast growing tumors without overt adverse effects on the major organs. GroEL is therefore a

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promising drug delivery platform that can overcome the obstacles to hydrophobic drug targeting and delivery. Keywords: Chaperonin, GroEL, Nanocarrier, ATP excitation, Hydrophobic drug delivery, Tumor targeting

The effective and selective delivery of hydrophobic therapeutic drugs, especially those targeted to tumors, is one of the key challenges facing drug discovery research and pharmaceutical development1-4. The use of a natural protein as drug delivery vehicle has been greatly successful in the case of albumin5. Some cage-like proteins are also being used as drug delivery vehicles, but often perform poorly because they lack a controlled drug release mechanism6, 7. GroEL, a molecular chaperone belonging to the chaperonin family, was previously employed as a biomolecular robotics, fueled by intracellular ATP in vitro8. GroEL, has a double layer cage structure, which acts as a hydrophobic environment for the folding of its substrate proteins. The chamber could be used as a hydrophobic drug container. In addition, GroEL can be excited by ATP molecules to release the folded substrates, suggesting that the loaded drug molecules could be released in a similar manner. In the present study, we explored the use of GroEL as a novel and intelligent hydrophobic drug delivery molecular machine. We loaded the hydrophobic antitumor drug, Doxorubicin (Dox), a first line chemotherapeutic drug, into GroEL (GroEL-Dox) with the goal of effectively and specifically treating tumor tissue. Our results prove that GroEL is able to load hydrophobic drugs without changing the overall protein structure, and effectively shield drugs from the aqueous phase of blood. Furthermore, we show that the loaded drugs can be released in the presence of a critical concentration of ATP at tumor sites. Most importantly, we found that GroEL specifically interacts with plectin, a cell structural protein that is abnormally expressed on the membranes of tumor cells but not in normal cells9, 10. This further augments the superior benefits of GroEL as a tumor-targeting carrier. The central dogma of targeted drug delivery is to steer therapeutic cargo to target tissues and/or cells to achieve maximal therapeutic efficacy with minimal toxic effects. There are two major strategies, from a materials approach, to design hydrophobic antitumor drug delivery systems. One is to use synthetic carriers, such as liposomes and polymer-based drug carriers3, 1113

. These can readily carry drugs via their hydrophobic core and protect them during the delivery 2


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process with their hydrophilic shell. However, the stability of synthetic carriers is generally difficult to control and their rapid clearance, via the reticuloendothelial system (RES), is often problematic. Importantly, these carriers normally do not possess the properties required for tumor cell targeting and controlled drug release, often failing to meet clinical expectations for safety and efficacy. The second strategy is to exploit natural biological compartments or organisms, ranging from organelles to pathogens to even mammalian cells. In fact, viruses, bacteria and red blood cells (RBCs) all possess one or more features desirable in drug delivery carriers. However, the preparation of pathogens and RBCs is relatively difficult and often incompatible with hydrophobic antitumor drugs. Furthermore, these approaches usually lack defined drug release control mechanisms14-16. GroEL, a natural molecular machine17, 18, is the most abundant protein folding apparatus in prokaryotic cells, with excellent structural stability, and heat and acid resistance. GroEL comprises 14 subunits to form two symmetrical rings (a cis- and a trans-ring), each with 7 subunits, to form two functional cavities19. In its native structure, GroEL has two symmetrical hydrophobic cavities, providing a favorable environment for the stable binding of hydrophobic drugs. In Figure 1A (upper row), we illustrate the process of incorporating Dox into the cavity of GroEL. Hydrophobic molecules tend to enter and remain stable within a hydrophobic cavity20, 21. Thus, Dox is sequestered within the hydrophobic cavity of GroEL, and can be easily transported in blood circulation due to the high water solubility of GroEL. To generate the drug delivery system, we loaded GroEL by incubating it with Dox for about 24 h at 37°C. The hydrophobic Dox molecules, which can be detected by UV-Vis spectroscopy, integrated into the cavity of GroEL (Figure 1B). Importantly, incorporation of Dox into GroEL does not change the protein’s overall architecture and integrity. As shown in Figure 1C-D, the Cryo-EM images of GroEL and GroEL-Dox indicate that the shape and size of GroEL-Dox are the same as GroEL alone. However, the UV-Vis spectrum of GroEL-Dox was markedly different from that of GroEL; GroEL-Dox has an additional peak at 509 nm, attributed to Dox (Figure 1B). Compared with other synthetic nanocarriers, such as peptide nanoparticles (maximal encapsulation efficiency up to 98%)22 and polymeric nanoparticles (up to 96%)23, the natural protein GroEL has a similar drug encapsulation efficiency under the optimal conditions (Table S1). GroEL is an ATPase24. Assembly of the functional ring of GroEL is coordinated by the activity of the 14 subunits, driven by the hydrolysis of ATP, while ATP binding will cause a 3


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conformational switch, resulting in a transition of the inner surface of the cavities from a hydrophobic to a hydrophilic state17, 25-29. Because of the high metabolic rate of tumor cells, the concentration of ATP in the tumor interstitium is consistently elevated (in the level of several hundred micromolar), compared to the barely detectable levels in healthy tissue30, 31. Thus, the high concentration of ATP within the tumor microenvironment can trigger GroEL to undergo a conformational switch and a subsequent release of Dox at the tumor sites. This metabolic statedependent release of drug may help minimize the off-target effects of drugs in healthy tissues. Based on this hypothesis, we first tested the effect of ATP on Dox release from GroEL-Dox in vitro. Figure 1A (lower row) summarizes our working model of ATP-triggered Dox release from GroEL-Dox. Six steps are required for the complete release of Dox from GroEL’s cavity: 1) ATP binds to the cis-ring of GroEL-Dox, causing the cavity inner face to begin changing from a hydrophobic to a hydrophilic state. Dox molecules begin to be released; 2) the cavity of the cisring becomes completely hydrophilic and the remaining Dox molecules are forced out of the ring; 3) ATP is hydrolyzed to ADP; 4) ATP bound to the trans-ring of GroEL begins a process analogous to Step 1; 5) the second ring becomes fully hydrophilic, releasing the remaining Dox molecules; 6) ATP is hydrolyzed. When Dox is excited by orange light (508 nm), it emits a red fluorescence32, 33. However, when fluorescent molecules are tightly packed together, their fluorescence signals can be quenched34-36. According to this principle, we expected that when Dox is released from the cavity of GroEL, we would observe an increase in fluorescence. We first tested this by treating GroEL-Dox with SDS (sodium dodecyl sulfate). SDS can completely denature GroEL37, 38 to release all of the Dox cargo. DNA molecules were also added to bind the released Dox, keeping the hydrophobic molecules from precipitating (Figure 1E). The increased fluorescence intensity of GroEL-Dox+DNA+SDS, compared to GroEL-Dox, was used as a fluorescence standard to evaluate the efficiency of Dox release when ATP was added into a GroEL-Dox+DNA solution. We used a concentration of ATP at 350 µM, as 700 µM is the upper limit of ATP concentration reported in the tumor microenvironment31. As shown in Figure 1F, after about 3 h incubation of the GroEL-Dox with DNA and ATP, the cumulative Dox release reached more than 80%, while the control group, without ATP, was less than 20%. This in vitro Dox release experiment demonstrates that GroEL-Dox can be triggered to efficiently release Dox by ATP concentrations found in the tumor microenvironment.

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To better address generality and practicability of the delivery system, we loaded another hydrophobic drug paclitaxel into GroEL. The GroEL-Paclitaxel was obtained using the same drug loading procedure for Dox. For drug release study, 350 µM ATP was added to trigger paclitaxel release from GroEL-Paclitaxel. GroELs before and after drug loading were measured by electrophoresis. The gel shift assay showed that the molecular weight of GroEL changed as expected, after loading and release of paclitaxel (Figure S1). The results demonstrate that the hydrophobic paclitaxel can be successfully loaded into GroEL to become a component of GroEL-Paclitaxel, which had a higher molecular weight (running slower in the gel shift assay). And the molecular weight of GroEL-Paclitaxel reduced after paclitaxel was released from GroEL, triggered by ATP hydrolysis. As shown in Table S2, the encapsulation efficiency of GroELPaclitaxel was up to 92%. In the course of our experiments, we were surprised to find that GroEL appeared to exhibit specificity for tumor cells, so we further examined whether protein(s) on the tumor cell membrane may serve as (a) receptor(s) or binding protein(s) for GroEL. We first extracted the total membrane proteins from the pancreatic cancer cell line, Panc-1. We then linked avidin to GroEL molecules and immobilized them onto streptavidin-coupled dynabeads. The Panc-1 membrane protein fraction was then incubated with the dynabeads. We finally eluted the proteins specifically binding to the beads and resolved them by SDS-PAGE (Figure 2A). The resulting protein gel reveals additional protein bands. We further characterized these candidate proteins by quantitative proteomic analysis. The proteins with the four highest scores were identified as the cellular structural protein, plectin, and its isoforms (Table S3). Bidirectional immunoprecipitation experiments of GroEL and plectin were carried out to verify that the two molecules indeed undergo a specific intermolecular interaction (Figure 2B-C). Our results show that, in both IP experiments, GroEL and plectin bind specifically and tightly, as each of the pair can be pulled down using an antibody specific for the respective partner. We next examined the levels of plectin in isolated membrane fractions from two cancer cell lines and two nonmalignant cell lines (human breast cancer cell line, MDA-MB-231; pancreatic cancer cell line, Panc-1; pancreatic normal epithelial cell line, HPNE; and human embryonic kidney cells, 293T). The expression of plectin in the membranes from the tumor lines was significantly higher than that in the two non-tumorous cell lines (Figure 2D). Furthermore, the

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uptake of GroEL in live cell experiments revealed that, after blocking nonspecific binding sites with goat serum, GroEL can still bind to the cell membranes of Panc-1 cells at 4°C. When the competing plectin antibodies were added in excess, the binding of GroEL was markedly reduced (Figure 2E). To further examine the binding of GroEL to plectin, we conducted the immunofluorescence assay (Figure S2). Since plectin is a cellular structural protein, the red fluorescence representing plectin was distributed within the whole cells. The green fluorescence representing GroEL in Panc-1 cells mainly localized on cell membrane. The merged image showed that GroEL and plectin were co-localized on the cell membrane. The overlap coefficient was calculated as approximately 0.98 and the Pearson’s R value was 0.39 (ImageJ software). As a negative control, HaCat cells showed no GroEL binding to the cell membrane. These data demonstrate that GroEL specifically binds to tumor cell membranes that possess an elevated plectin presence. To examine the cellular uptake and drug release profile of GroEL-Dox, we incubated MDAMB-231 cells with 5-carboxyfluorescein N-succinimidyl ester -labeled GroEL (FAM-GroEL) at room temperature. After 1 h incubation, the fluorescent green, FAM signal began accumulating around the surface of the MDA-MB-231 cells. The signal was exclusively on the surface of the cells for at least 6 h and did not overlap with red fluorescence from LysoTracker-stained lysosomal compartments within the cells (Figure 3A), indicating that GroEL binds to the MDAMB-231 cell membrane, without entering the cell. This membrane localization may provide the additional advantage of escaping the deleterious effects of lysosomes, which is where conventional drug carriers often accumulate, entering cells through endocytosis. The endosomes mature into inhospitable, lysosomal compartments, where the agents may be degraded or otherwise inactivated39. We next investigated the ability of GroEL-Dox to release the drug into cells. After 1 h incubation, the fluorescent signal (green) from FAM-GroEL-Dox began accumulating on the membrane of MDA-MB-231 cells. However, after 6 h, a red signal from the released Dox appeared both at the plasma membrane and throughout the cytosol (Figure 3B). The exclusion of the green signal from the inside of cells suggests that Dox was released outside the cells, followed by a highly efficient, hydrophobic drug penetration of the cell membrane. To examine whether GroEL can efficiently and specifically target tumor tissue in vivo, we employed an animal tumor model. BALB/c nude mice were implanted with MDA-MB-231 human breast tumors which were allowed to grow for 2 weeks before the administration of 6


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Cy5.5-labeled GroEL (GroEL-Cy5.5) into the tail vein of mice. In vivo, whole-body, nearinfrared fluorescence imaging revealed an enrichment of the fluorescent signal in the tumor tissue. This enrichment was highly efficient, as 95% of the signal was localized to the tumor tissue (Figure 3C, red). To further confirm the in vivo imaging results, various organs were excised for ex vivo imaging (Figure 3D). Under the same fluorescent excitation conditions, about 95% of the fluorescent signal was associated with the excised tumor tissue. These data show that GroEL has an exceptional tumor-targeting ability both at the cellular and live animal levels. Importantly, the tumor killing effect of GroEL-Dox achieved satisfactory results in MDAMB-231 tumor models (Figure 3E). To determine the ATP concentration in breast tumor model, we established MDA-MB-231 tumor xenograft in nude mice. Mice were sacrificed when tumor volume reached about 100 mm3. The main organs and tumors were immediately excised for subsequent detection of ATP levels, by a ATP assay kit (S0027, Beyotime). The results showed that the concentrations of ATP in normal tissues varied from 5-100 µmol/mg protein; while in MDA-MB-231 xenograft tumor tissues, the concentration of ATP could reach approximately 400 µmol/mg protein (Figure S3). To study the bloodstream clearance of GroEL, we carried out additional pharmacokinetics analysis of GroEL in BALB/c nude mice. Mice were administrated with 100 µL Cy7 labeled GroEL (GroEL-Cy7) or 100 µL free Cy7 (as a control) via tail vein, respectively. At various time points after injection, 20 µL blood was withdrawn from a tail cut. Then all blood samples were subjected to fluorescent imaging. The results showed that the GroEL-Cy7 molecules have an apparent half-life approximately 6 h and more than 40% of GroEL fluorescent signal was still detectable in the blood 12 h post-injection; while free fluorescent dye was cleared quickly (Figure S4). To examine the therapeutic potential of GroEL-Dox, we used BALB/c nude mice bearing Panc-1 human pancreatic tumors (Figure 4), implanted 2 weeks prior the treatment40. The tumor bearing mice were randomized into four different treatment groups (n = 6 / group). When the tumor volume reached 100 mm3, we treated each group with HBS, GroEL, free Dox or GroELDox at a Dox dosage of 2 mg/kg (for the two groups with a Dox formulation) by intravenous injection. The procedure was repeated once every 2 days, seven times. The tumor volumes and body weights were recorded. After the 12-day therapeutic period, the GroEL-Dox group 7


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exhibited a significantly enhanced antitumor effect (Figure 4A) with the final tumor weight down to nearly one third that of the vehicle-treated control (Figure 4B-C). Importantly, none of the treatments affected the animals’ body weights (Figure 4D). To evaluate whether apoptosis was the cause of the antitumor effects, tumor sections were subjected to terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining analysis. The GroEL-Dox treated groups showed a widespread tumor cell apoptosis, compared to the control groups (Figure 4E). GroEL has previously been shown to be highly immunogenic41, so it is important to evaluate the safety of GroEL-based reagents. To investigate this, the heart, liver, spleen, lung and kidney of mice bearing Panc-1 human pancreatic tumors and treated with GroEL-Dox were sectioned and examined by H&E staining. Paraffin sections of these main organs were similar in all groups, with no apparent additional damage or abnormal histological alterations observed in GroEL-Dox treated group compared to the control group, including no inflammatory infiltrates into the tissues, indicating a general safety of GroEL-Dox to the major organs (Figure 4F). We also examined biochemical markers of hepatic and renal function in the serum of treated mice (aspartate transaminase, AST; alanine aminotransferase, ALT; blood urea nitrogen, BUN; serum creatinine, Scr) (Figure S6). GroEL had no obvious impact on these markers, while treatment with free Dox resulted in a significant increase in the serum AST level, indicating liver injury. Importantly, the AST level in GroEL-Dox group was within the normal range. Thus we conclude that GroEL itself has minimal side effects for liver and kidney, and GroEL can protect the liver toxicity of Dox. Furthermore, the general health of the mice was not affected by any of the treatments and the animals showed no loss in weight, indicating that they were eating normally. To further confirm the therapeutic efficacy of GroEL, the MDA-MB-231 breast tumor model was used. After an 18-day treatment period, GroEL-Dox also showed a significantly enhanced antitumor effect (Figure S5A) and the final tumor weights were significantly lower than those of the control group (Figure S5B). Furthermore, the heart, liver, spleen, lung, kidney and tumors were sectioned and examined by H&E staining analysis in the MDA-MB-231 model. Again, no obvious tissue damage or inflammatory infiltrates were observed. As expected, significant tumor cell damage was observed in GroEL-Dox treated group, but not the control group (Figure S5C). To investigate the immune responses to GroEL, 2 mg/kg GroEL or lipopolysaccharide (LPS, as a positive control) were intravenously injected into the BALB/c 8


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mice, and the serum levels of IFN-γ, IL-2 and IL-6 were measured by ELISA after 6 h and 24 h treatment. As showed in Figure S7, GroEL slightly increased the serum levels of IFN-γ, IL-2 and IL-6, indicating GroEL provoked limited immune responses. Importantly, this increase was much lower than that of LPS treatment. Although further more detailed immunotoxicity studies are required, our data show that the dose of GroEL used in the current study can only provoke limited immune responses and the serum levels of IFN-γ return to normal within 24 h. Taken together, our results indicate that GroEL-Dox possesses a high antitumor efficacy with few side effects. We propose a working model (Figure 5) of GroEL-Dox for drug delivery and release specifically to tumor cells. GroEL-Dox first enters into the tumor vasculature, where the drug can penetrate the tumor blood vessel wall through the abnormal vasculature structure and openings. Once inside the tumor interstitium, GroEL-Dox specifically binds to tumor cell membranes through the target protein plectin. Due to the presence of higher concentration of ATP in the tumor microenvironment, the ATP triggers a conformational switch in GroEL-Dox to release Dox. The released free hydrophobic Dox molecules pass though the plasma membrane lipid bilayer into the cell and eventually enter the nucleus, where the drug binds to DNA, blocking replication and causing apoptosis. It is highly likely that GroEL only binds to the cell membrane, but does not enter into tumor cells. This is a desirable property which allows the escape from lysosomes In summary, we have exploited a naturally occurring protein as an intelligent, tumortargeting, hydrophobic antitumor drug delivery molecular machine. In our experiments, GroELDox exhibited significant tumor killing efficacy at both the cellular and organismal levels. In the process of drug delivery, GroEL can protect the hydrophobic drug Dox from undesired degradation in the bloodstream until the GroEL-Dox reaches the tumor site, where it releases the drug when triggered by the high concentration of ATP within the tumor microenvironment. Our results suggest that GroEL only binds to tumor cell membranes, remaining outside the cells, thus escaping the deleterious effects of lysosomes, whose acidic conditions often interfere with encapsulated drug-delivery. GroEL specifically interacts with tumor cells; at least one of its target partners may be plectin, which is expressed at abnormally high levels on the membranes of tumor cells. Finally, GroEL possesses low or no cytotoxicity in mammalian systems.

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Considering the excellent antitumor effects we observed in live animals, we believe that GroEL, and possibly other chaperonin family members with similar structures and molecular mechanisms, are excellent candidates as hydrophobic antitumor drug delivery systems. Further studies with these naturally occurring protein complexes for hydrophobic drug delivery may alleviate the obstacles and cost of hydrophobic drug modification, opening a door for clinical development of this class of natural protein cage-drug delivery vehicles.

ASSOCIATED CONTENT Supporting Information. Experimental materials and methods, encapsulation efficiency, quantitative proteomic analysis, immunofluorescence, ATP concentrations, pharmacokinetics, anti-tumor effect in vivo, liver and renal functions and immune responses are presented in the Supporting Information. This material is available free of charge via the Internet at hppt://pubs.acs.org.

Acknowledgments: This work was supported by the National Natural Science Foundation of China (31500814, 31730032, 21373067 and 51673051), the National Distinguished Young Scientists program (31325010), the Innovation Research Group of the National Natural Science Foundation (11621505), the Key Research Project of Frontier Science of the Chinese Academy of Sciences (QYZDJ-SSW-SLH022), , and Beijing Municipal Science & Technology Commission (Z161100000116035). We thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) for the use of Cryo-EM facility. This work was also supported by Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, Chinese Academy of Sciences and K.C.WONG Foundation, CAS (Y7552911ZX).

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Figure 1. Assembly and in vitro drug release of GroEL-Dox, and characterization of the GroELDox complex. (A) Schematic illustration of the assembly of GroEL-Dox and in vitro Dox release from GroEL-Dox, triggered by ATP. ATP combines with the cis-ring and trans-ring of GroEL causing a conformational change in the chaperonin to release the Dox cargo. (B) The UV-Vis spectra of GroEL-Dox and GroEL. The inset shows the color difference between GroEL solution and GroEL-Dox. (C-D) Cryo-EM images of GroEL (C) and GroEL-Dox (D), Scale bar = 20 nm. The insets show the top or side views of individual GroEL molecules at a higher magnification (75000×). (E) The fluorescence intensity changes of Dox after addition of SDS and DNA to GroEL-Dox to denature GroEL and release of Dox molecules. (F) In vitro Dox release profile of GroEL-Dox when triggered by ATP hydrolysis. ATP was added to the solution of GroEL-DoxDNA at a concentration of ATP of 350 µM.

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Figure 2. Specific interaction between GroEL and plectin. (A) SDS-PAGE analysis of the magnetically precipitated proteins and total membrane proteins of Panc-1 cells and GroEL samples. The protein bands of the eluate and pure GroEL samples were compared; the additional protein bands in the precipitated complex are labeled with red asterisks. (B) Immunoprecipitation of plectin by Protein G-anti-GroEL-GroEL. (C) Immunoprecipitation of GroEL by Protein Ganti-plectin-plectin. (D) Plectin levels in the membrane fractions from 2 tumor cell lines (MDAMB-231 and Panc-1) and 2 non-tumor cell lines (HPNE and 293T). (E) Association of GroEL with Panc-1 cells and competitive inhibition by anti-plectin antibodies.

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Figure 3. Subcellular distribution, in vitro Dox release and in vivo distribution of GroEL. (A) Subcellular GroEL distribution in MDA-MB-231 cells. 50 nM FAM -GroEL was added to MDA-MB-231 cells. The distribution of the fluorescence was examined by confocal microscopy at the indicated times. Green, FAM- GroEL; red, LysoTracker. Scale bar = 50 µm. (B) In vitro Dox release of GroEL-Dox into MDA-MB-231 cells, treated with a fluorescence-labeled version of the complex (FAM-GroEL-Dox). Scale bar = 50 µm. (C) In vivo GroEL distribution in nude mice bearing MDA-MB-231 human breast tumors. The tumor regions are marked by dotted lines. (D) Ex vivo images of fluorescent GroEL in organs and tumors of nude mice bearing MDA-MB231 human breast tumors. Red, GroEL-Cy5.5; green, background fluorescence. (E) MDA-MB231 cell viability and cytotoxicity assays after treatment with GroEL-Dox, GroEL, Dox or HBS.

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Figure 4. Enhanced antitumor effects of GroEL-Dox in vivo. (A) Tumor growth curves of the different experimental groups after treatment with the indicated substances. Nude mice bearing Panc-1 human pancreatic tumors, implanted 2 weeks prior, received intravenous injections of GroEL-Dox (2 mg/kg), Dox (2 mg/kg), or HBS every other day. ** p < 0.01. (B) The tumors were harvested and weighed after 12 days of treatment. n = 6 for each group in (A) and (B). * p < 0.05, ** p < 0.01, ns = no significance. (C) The tumors were measured after a 12-day therapeutic period; all mice were sacrificed. (D) The body weights were recorded every 2 days during the treatment. (E) The tumor sections were examined by TUNEL staining analysis. Scale bar = 100 µm. (F) H&E stained paraffin sections of a panel of organs from the mice sacrificed in the groups shown in D. Scale bar = 100 µm.

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Figure 5. Proposed mechanism of action of GroEL-Dox in drug delivery and release to tumor cells. When GroEL-Dox is administered intravenously to nude mice, the aqueous solubility of GroEL permits the transport of the hydrophobic drug, Dox, safely through the circulation. Once inside the tumor vasculature, GroEL-Dox penetrates through the vessel wall and enters the tumor microenvironment. In the tumor microenvironment, the high concentration of ATP leads to: 1) GroEL-Dox binding to its target partner plectin, which is highly expressed on tumor cell membranes; 2) the triggering of a conformational change in GroEL, leading to a shift to hydrophilicity in the protein cavity; 3) Dox is released from the cavity of GroEL-Dox, where it then readily penetrates the lipophilic tumor cell membrane eventually entering the nucleus and causing apoptosis.

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Graphic Table of Contents Chaperonin-GroEL as a smart hydrophobic drug delivery and tumor targeting molecular machine for tumor therapy

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Chaperonin-GroEL as a smart hydrophobic drug delivery and tumor

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