Letter pubs.acs.org/NanoLett
Hyaluronidase To Enhance Nanoparticle-Based Photodynamic Tumor Therapy Hua Gong, Yu Chao, Jian Xiang, Xiao Han, Guosheng Song, Liangzhu Feng, Jingjing Liu, Guangbao Yang, Qian Chen, and Zhuang Liu* Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123, China
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ABSTRACT: Photodynamic therapy (PDT) is considered as a safe and selective way to treat a wide range of cancers as well as nononcological disorders. However, as oxygen is required in the process of PDT, the hypoxic tumor microenvironment has largely limited the efficacy of PDT to treat tumors especially those with relatively large sizes. To this end, we uncover that hyaluronidase (HAase), which breaks down hyaluronan, a major component of extracellular matrix (ECM) in tumors, would be able to enhance the efficacy of nanoparticle-based PDT for in vivo cancer treatment. It is found that the administration of HAase would lead to the increase of tumor vessel densities and effective vascular areas, resulting in increased perfusion inside the tumor. As a result, the tumor uptake of nanomicelles covalently linked with chlorine e6 (NM-Ce6) would be increased by ∼2 folds due to the improved “enhanced permeability and retention” (EPR) effect, while the tumor oxygenation level also shows a remarkable increase, effectively relieving the hypoxia state inside the tumor. Those effects taken together offer significant benefits in greatly improving the efficacy of PDT delivered by nanoparticles. Taking advantage of the effective migration of HAase from the primary tumor to its drainage sentinel lymph nodes (SLNs), we further demonstrate that this strategy would be helpful to the treatment of metastatic lymph nodes by nanoparticlebased PDT. Lastly, both enhanced EPR effect of NM-Ce6 and relieved hypoxia state of tumor are also observed after systemic injection of modified HAase, proving its potential for clinical translation. Therefore, our work presents a new concept to improve the efficacy of nanomedicine by modulating the tumor microenvironment. KEYWORDS: Hyaluronidase, photodynamic therapy, nanomedicine, tumor microenvironment, lymphatic metastasis
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oxygen supply to the tumor, particularly tumor cells far from blood vessels, often lead to the hypoxia-associated resistance of PDT for many types of cancers.10−12 In recent years, a number of groups have tried to modify the tumor microenvironment so as to create better conditions for cancer treatment.13−15 Rakesh et al. proposed a novel rationale that normalization of tumor vasculatures but not destructing them by using antiangiogenesis drugs could facilitate the penetration of drugs and oxygen into tumor tissues in 2001.16 Subsequently, normalization of tumor vasculatures has been successfully combined with chemotherapy,17−20 radiotherapy,20−22 as well as PDT.23−25 Different from the above strategies for normalization of tumor blood vessels, directly lowering the interstitial flow pressure (IFP) by decomposing the extracellular matrix (ECM) might be another alternative way.26 Hyaluronan (HA), which is constituted of repeating units of N-acetylglucosamine and glucuronic acid disaccharide,
hotodynamic therapy (PDT), which relies on light excitation of photosensitizing molecules to generate singlet oxygen (SO) or reactive oxygen species (ROS) from oxygen molecules, has been proven to be a safe and selective method to cure a wide spectrum of cancers and nononcological disorders in the clinic.1,2 Because both photosensitizer (PS) and oxygen determine the final therapeutic effects of PDT, the local concentration of PS molecules in tumors and oxygen contents inside the tumor microenvironment are important decisive factors for PDT.3−5 To enhance the tumor homing of PS molecules, nanomedicine approaches by using PS-loaded nanoparticles have been developed to improve the pharmacokinetics and tumor retention of PS molecules, mostly relying on the enhanced penetration and retention (EPR) effect of cancerous tumors in which the blood vessels are tortuous and leaky.6,7 However, for early stage metastatic tumors as well as the central part of large tumors, their vascular network might be not abundant, and thus the intratumoral blood perfusion could be relatively low, leading to the largely weakened EPR effect in those tumors.8,9 On the other hand, the rapid consummation of oxygen by fast-growing tumor cells, together with inefficient © 2016 American Chemical Society
Received: January 6, 2016 Revised: February 29, 2016 Published: March 29, 2016 2512
DOI: 10.1021/acs.nanolett.6b00068 Nano Lett. 2016, 16, 2512−2521
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Nano Letters is a key component of extracellular matrix (ECM).27,28 The major function of HA is to provide a hydrated gel-like matrix to support the tumor growth.29−31 A wide range of human tumors have been reported to show high expression of HA, whose level is positively correlated with the tumor grade, possibility of distal metastasis, and overall survival.30,32,33 Hyaluronidase (HAase), which functions as an enzyme to break down HA at specific sites, has been used as an adjuvant for chemotherapy to enhance the penetration of drugs for many years.34−36 However, whether the introduction of HAase would enhance the efficacy of PDT remains to be explored to our best knowledge. In this work, we have carefully studied how administration with HAase would affect the vasculatures, blood perfusion, and oxygenation in the tumor and tried to combine HAase treatment with nanoparticle-based PDT for synergistic cancer therapy. It was found that both the tumor vascular densities and effective vasculature areas were increased after HAase administration, inducing enhanced perfusion inside the tumor. As a consequence, the hypoxia state inside the tumor treated with HAase was dramatically relieved compared with salinetreated tumors. In the meantime, owing to the enhanced tumor EPR effect after HAase treatment, the tumor uptake of nanomicelles covalently linked with chlorine e6 (NM-Ce6), a photosensitizer, was enhanced by ∼2-fold. Such an increased tumor homing of PS molecules together with the improved tumor oxygenation resulted in remarkably enhanced PDT treatment efficacy in a mouse tumor model (Scheme 1). Beyond the treatment of subcutaneous tumors, we further uncovered the enhanced EPR effect for nanomicelles and
alleviated hypoxia state in the metastatic sentinel lymph nodes (SLNs) after injection of HAase into primary tumors. Moreover, it was also demonstrated that systemic injection of polyethylene glycol (PEG)-modified HAase could also help to enhance the EPR effect of NM-Ce6 and relieve the local hypoxia in the tumor. Therefore, such a strategy may indeed be a helpful supportive method to promote cancer PDT treatment with substantial potential for clinical translation. Results and Discussion. 1. HAase Treatment Could Increase the Density of Tumor Blood Vessels and Effective Vascular Areas. The disruption of ECM in the tumor would result in decreased IFP, which has direct impacts on the physiologic state of tumor vasculatures.37 Therefore, we first wondered whether HAase treatment could lead to the normalization of tumor vasculatures and enhance the tumor perfusion. By using dye-labeled anti-CD31 antibody, the immunofluorescence images of tumor blood vasculatures were obtained for frozen cross-section slices of tumors. By analyzing the interior, middle, and exterior regions of tumor slices in six separated layers, both the density and the physiologic state of tumor vasculatures were analyzed. It was found that the blood vessel density in the tumor after HAase treatment for 24 h was dramatically enhanced (Figure 1a,b). Furthermore, the percentage of dilated blood vessels as characterized by the complete margins was much higher after HAase treatment than that in the saline treated group (white arrows indicate dilated blood vessels), indicating the increase of effective tumor vasculature area induced by HAase (Figure 1a,c). It is known that the increase of both vasculature density and effective tumor vasculature area would contribute to the enhanced tumor perfusion.38 To confirm this, ultrasound imaging with a commercial microbubble contrast agent was used to study the blood perfusion inside tumors. Compared to untreated tumors in which only surrounding regions were filled with contrast agents (Supplementary Video 1), strong ultrasound signals quickly showed up right after injection of microbubbles in most regions of HAase treated tumors (Supplementary Video 2), indicating the greatly enhanced tumor perfusion after HAase treatment (Figure 1d). Both ex vivo immunofluorescence imaging of tumor slices and in vivo ultrasound imaging of tumors revealed that the HAase treatment could help to normalize the tumoral vasculature function, and thus enhance the tumor perfusion. Probably owing to the breakdown of tumor ECM, the IFP in the tumor region would decrease, resulting in the dilation of previously compressed vessels. Though the tumor vascular network is no less abundant than that in normal tissues, a large majority of tumor vessels are compressed and nonfunctional due to the increased intratumoral IFP.26,39,40 Therefore, the injection of HAase could make those blood vessels become functional again. Furthermore, the oligosaccharide products hydrolyzed from HA after HAase treatment could serve as an angiogenesis mediator,41,42 which may facilitate the vessel growth in TME. To sum up, after HAase treatment, both the increased vessel density and enlarged effective vascular areas contribute to the enhanced perfusion inside the tumor. 2. HAase Treatment Could Enhance the Tumor EPR Effect for Nanoparticles. With the enhanced tumor perfusion after HAase treatment, we wondered whether the administration of HAase could result in the enhanced EPR effect of nanoparticles. PEGylated, Chlorine e6 (Ce6)-coupled poly(maleic anhydridealt-1-octadecene), a PS-conjugated amphiphilic polymer reported previously,43 would form Ce6-containing nanomicelles
Scheme 1. Schemes Showing the Effects of HAase on the Modulation of Tumor Microenvironment, Resulting in Enhanced Efficacy of in Vivo PDT Cancer Treatment via Improving Tumor Oxygenation and Promoting EPR Effect for Nanoparticles
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Figure 1. Intratumoral injection of HAase increased the tumor perfusion. (a) Representative fluorescence images of tumor blood vessels of frozen tumor slices stained by anti-CD31 after treating with 1500 U HAase or saline. (b) Statistic data of vascular pixel area (red) percentage of slices obtained from the exterior, middle, and interior regions of six representative layers after treating the tumor with 1500 U HAase or saline. (c) Statistic data of effective vascular area characterized by dilated vascular cross section of the above selected slices. The data in (b,c) were analyzed through Image-Pro Plus 6.0. Student’s t test was used. (* indicates p < 0.05, and ** indicates p < 0.01) (d) Ultrasound images of tumor vascular perfusion after treating the tumor with 1500 U HAase or saline (also see Supplementary videos). Microbubbles were intravenously injected as the ultrasound contrast agent.
increased in the kidney and spleen after HAase administration into the tumor, although the exact reason remains to be unclear. HAase has been used as an adjuvant for chemotherapy for many years to enhance tumor uptake of small molecular drugs and nanodrugs.32,35,36,45 The possible mechanism for the enhanced EPR effect could be attributed to the lowered IFP owing to the breakdown of HA molecules in the extracellular matrix of tumor, as well as the improved tumor perfusion as shown in Figure 1.46,47 3. HAase Treatment Could Dramatically Relieve the Hypoxia State Inside the Tumor. Inspired by the enhanced tumor perfusion after HAase treatment, it is reasonable to predict that the hypoxic state of the tumor might be greatly alleviated after HAase treatment. Therefore, we used Pimonidazole, a hypoxia-specific probe that binds with thiolcontaining proteins in hypoxic cells,48 to detect the tumor hypoxia state. Antimouse CD31 antibody was used at the same time to locate blood vessels inside the tumor. For untreated tumors, large hypoxic areas were noticed in exterior, middle, and interior regions of a tumor (Figure 3). Interestingly, after treatment with HAase at the dose of 1500 U, the hypoxiastained signals became obviously lower within the whole tumor. Furthermore, we found that the hypoxic area was in a negative correlation with both the density and dilated state of blood vessels in the HAase treated tumor, indicating that the enhanced normalization of tumor blood vessels induced by HAase could be the critical reason that leads to the greatly relieved hypoxia status in the tumor (Figure 3). Besides Pimonidazole-based hypoxia staining, antihypoxia inducible factor 1 alpha (HIF-1α) antibody staining was also used for evaluation of the tumor hypoxia state. HIF-1 α is an oxygen regulated subunit of HIF-1, and its existence is highly determined by the local oxygen level. While being rapidly
(NM-Ce6) in the aqueous solution (Figure 2a). Spherical nanoparticles with uniform sizes were observed under transmission electron microscopy (TEM) after staining those micelles with phosphotungstic acid (Figure 2b). The average hydrodynamic diameter of NM-Ce6 was measured by dynamic light scattering (DLS) to be about 33 nm (Figure 2c). The characteristic Ce6 peaks were noticed in the absorption and emission spectra of NM-Ce6, confirming the successful loading of Ce6 in those nanoparticles (Figure S1a,b). We next studied how HAase administration would affect the tumor uptake of NM-Ce6 nanoparticles. HAase with different doses was directly injected into one of two 4T1 tumors growing on each Balb/c mouse, which was then intravenously (iv) injected with NM-Ce6 (200 μL, 1.0 mg/mL of Ce6, dose = 10 mg/kg). In vivo fluorescence imaging was conducted for those mice by recording Ce6 fluorescence. Compared to salineinjected tumors, tumors injected with 1500 U of HAase showed the most obviously enhanced tumor uptake of NM-Ce6 (Figure 2d,e). We thus chose 1500 U of HAase as the optimal dose in our followed experiments. To quantitatively determine the enhanced EPR effect by HAase treatment, we used radioisotope labeled NM-Ce6 for biodistribution measurement. Chlorin e6 with a porphyrin ring structure could be used as a chelator for metal irons, such as Gd3+and Mn2+.43,44 Inspired by this, we used Ce6 to chelate 99m Tc4+ (Figure 2a), whose γ-ray emission could be used to track the biodistribution of NM-Ce6-99mTc through a γcounter. On the basis of the quantitative biodistribution measurement of 99mTc-labeled NM-Ce6, we found that that uptake of nanoparticles in tumors treated with HAase was ∼2folds of that in the saline-injected group (Figure 2e), indicating that HAase treatment could indeed enhance the tumor EPR effect for nanoparticles. The uptake of NM-Ce6 also slightly 2514
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Figure 2. HAase treatment enhanced the tumor EPR effect. (a) A scheme showing the structure of NM-Ce6. Ce6 could be used as chelators for 99m Tc4+. (b) TEM images of NM-Ce6 negatively stained by phosphotungstic acid. (c) DLS date of NM-Ce6 showing their average hydrodynamic diameter to be 33 nm. (d) In vivo fluorescence imaging of Balb/c mice bearing 4T1 tumors (two tumors per mouse) after injection of NM-Ce6. The left tumors were injected with saline, while the right tumors were injected with series concentrations of HAase. (e) Statistic histograms of fluorescence intensity of both left and right tumor regions. The data were based on triplicated experiments. (f) Biodistribution of NM-Ce6 labeled with 99mTc determined 24 h post injection. Student’s t test was used. (* indicates p < 0.05, and ** indicates p < 0.01).
degraded by proteasome under normoxia conditions, HIF-1 α could persist for a long time under hypoxia conditions.49,50 Comparing with that in the saline treated tumor, the HIF-1 α expression level in HAase injected tumors was dramatically decreased, suggesting their greatly relieved hypoxia conditions (Figure S2), consistent to the results obtained by Pimonidazole-based hypoxia staining. 4. HAase Treatment Could Enhance the Efficacy of in Vivo Photodynamic Therapy. The amount of PS molecules and local oxygen concentrations in the tumor are important factors for effective in vivo PDT. Inspired by the enhanced EPR effect of PS-loaded nanoparticles and relieved hypoxia status after HAase treatment, we wondered whether the in vivo PDT effect could be enhanced with the aid of HAase treatment. Balb/c mice bearing 4T1 tumors were randomly divided into four groups (n = 5), saline injection group, HAase treatment group, PDT group with NM-Ce6 (dose =10 mg/kg in terms of Ce6), and HAase treatment plus PDT with NM-Ce6. The mice in the last two PDT groups were exposed to a 660 nm light irradiation for 1 h at the power density of 2 mW/cm2. Although PDT treatment by iv injection of NM-Ce6 and light irradiation of
tumors could obviously delay the tumor growth at the early stage post treatment, the tumors in this group grew larger later on. Notably, although HAase treatment alone showed no effect to the tumor growth, it could remarkably enhance the PDT efficacy with NM-Ce6: the tumor growth was almost completely inhibited after HAase treatment plus NM-Ce6 induced PDT (Figure 4a−c). Neither body weight loss nor tissue damage was observed after such a treatment, indicating that the HAase-enhanced PDT was a mild method and rendered no obvious side effects to the animals (Figure 4d and Figure S3). To compare the extent of tissue damages for different treatment groups, tumors were harvested 24 h after treatment. Hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of tumor slices were both conducted to determine the tissue damage and cell apoptosis, respectively, after various treatments. While HAase treatment induced no obvious harm to the tumor, PDT treatment with MN-Ce6 under the currently used dose would induce partial cell damages and a moderate level of apoptosis to tumor cells. In 2515
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Figure 3. Immunofluorescence imaging of tumor slices after treatment with 1500 U HAase. Saline-injected tumor slices were used as the control. The tumor blood vessels (red) were stained with the anti-CD31 antibody, while the hypoxic regions (green) were stained with an antibody specifically bind to preinjected Pimonidazole, which was trapped in hypoxic tissues. The nuclei of cells (blue) were stained with DAPI. Obviously reduced hypoxia signals were observed for tumors post-HAase treatment.
pathway upon systemic iv injection.60 Therefore, enhancing the efficacy of lymph node targeting, especially for SLNs, is critically important. We thus hypothesize that HAase injected into primary tumors may also diffuse into their SLNs, and then change the microenvironment there and facilitate the nanoparticle-based PDT treatment efficacy on those lymph nodes. The 4T1 metastatic tumor model was built by injecting tumor cells into the foot pad according to the reported protocols.56 First, to confirm the migration of HAase from the primary tumor to their nearby SLNs after intratumoral injection, we used a near-infrared dye Cy5.5 to label HAase so as to track the translocation of Cy5.5-HAase after administration into mice (Figure 5a). It was found that HAase injected into the primary tumor would gradually move toward its nearby lymph nodes through lymphatic circulation. The fluorescence of Cy5.5 reached the peak value 1 h after injection and was retained in those lymph nodes later, indicating the efficient translocation and accumulation of HAase in the drainage lymph nodes of primary tumors into which HAase was initially injected. Subsequently, we would like to know whether the migrated HAase could alter the local microenvironment of metastatic lymph nodes and bring the same biologic effects as it does to the primary tumor. Twelve days after inoculation of tumor cells
contrast, the majority of tumor cells lost their normal morphology under H&E staining after treatment with both HAase and MN-Ce6-based PDT, which also resulted in large areas of tumor cell apoptosis as revealed by TUNEL staining (Figure 4e,f). Our results taken together demonstrate that the efficacy of nanoparticles-based PDT could be dramatically improved by modulating the tumor microenvironment with the treatment of HAase. 5. HAase Injection to Enhance PDT Treatment of Lymphatic Metastasis. Tumor metastasis is the major cause of cancer death. Developing methods to control and inhibit tumor metastasis would be of great importance to extend the survival of cancer patients.51 Sentinel lymph nodes (SLNs) are known to be the primary target organ for metastatic tumor cells in the early stage of tumor metastasis.52,53 Therefore, eradicating tumor metastasis in SLNs at the same time as rooting out the primary tumor could be an effective way to prevent further tumor metastasis.54,55 It has been well documented that nanoparticles could migrate from primary tumors into SLNs via the lymphatic drainage pathway.56−59 However, delivering nanodrugs to metastatic lymph nodes with high efficiency has been relatively less explored despite a recent work reporting that some nanoparticles with diameters smaller than 50 nm could enter SLNs through the transvascular 2516
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Figure 4. In vivo PDT effect enhanced by administration of HAase. (a) Tumor growth curve of mice after treated with saline, HAase alone, PDT alone, and PDT plus HAase at day 0 and day 6. (b) Mice in each group were sacrificed with tumors taken out and displayed. (c) Tumor weight data in different groups of mice post various treatments. (d) Body weight data of mice in different treatment groups. (e,f) Micrographs of HE stained (e) and TUNEL stained (f) tumor slices 1 day after the first treatment.
primary tumors could enhance the EPR effect in their metastatic SLNs (Figure 5c). In the meanwhile, the hypoxic state in the draining lymph nodes after HAase treatment of the primary tumor was also evaluated. Hypoxia-stained slices of SLNs showed that hypoxiastaining signals in the lymph nodes on the HAase treated side were obviously weaker than that in the nodes from the salinetreated side (Figure 5d), suggesting HAase administration into primary tumors could indeed help to relieve the hypoxic state of drainage SLNs. Notably, although the tumor blood vessel density in the metastatic lymph node showed no significant increase after injection of HAase into the primary tumor, we observed more effective blood vessels, which are characterized by the “open ring” structure under CD31 staining, in those lymph nodes after treating with HAase (Figure S4). This may possibly be the reason for relieved hypoxia in those lymph nodes. Considering the enhanced EPR effect as well as the relieved hypoxic state in the metastatic SLNs after HAase treatment of primary tumors, we then studied whether such treatment would facilitate PDT toward tumor cells spreading into lymph nodes.
into the two rear foot pads of each mouse, tumor metastasis could form on both sides of popliteal lymph nodes. Considering the larger tumor sizes in this experiment compared to that in the previous subcutaneous tumor model experiment, a high dose of HAase (3000 U HAase in 50 μL saline) was intratumorally injected into the left footpad tumor. The same volume of saline was injected into the right side as the control. Two hours later, NM-Ce6 (dose = 10 mg/kg in terms of Ce6) was then iv injected into those mice. In vivo fluorescence imaging showed that the fluorescence intensities of both the primary tumor and its drainage lymph node on the left side (with HAase injection) were much stronger than those on the right side without HAase treatment (Figure 5b), indicating that the EPR effect in both primary tumors and drainage lymph nodes were enhanced due to administration of HAase. Furthermore, by using 99m Tc to label NM-Ce6, we quantitatively measured the biodistribtion of nanoparticles and uncovered that the lymph accumulation of NM-Ce6-99mTc for the HAase treatment side was about 2.5 times compared to that for the saline treated side, suggesting HAase treatment of 2517
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Figure 5. HAase administration changed the microenvironment of tumor metastatic lymph nodes and enhanced the in vivo PDT toward them. (a) In vivo fluorescence imaging of mice with 4T1 tumors inoculated on their foot pads and metastasis occurred in the SLNs. Cy5.5-HAase was injected into the primary tumor prior to imaging. (b) In vivo fluorescence imaging for mice with primary tumors and metastatic lymph nodes on both sides, taken 24 h after intravenous injection of NM-Ce6. The left tumor at the footpad was intratumorally injected with 3000 U HAase, while the right tumor was injected with saline. SLNs from two sides were taken out for ex vivo fluorescence imaging (the bottom image). (c) Lymph node uptake of NM-Ce6-99mTc in metastatic lymph nodes with and without HAase treatment of their respective primary tumors. Student’s t test were used. (* indicates p < 0.05, and ** indicates p < 0.01). (d) Immunofluorescence imaging of metastatic lymph nodes with and without HAase treatment of their respective primary tumors. Tumor blood vessels, hypoxic regions, and cell nuclei were stained with anti-CD31 (red), antipimonidazole (green), and DAPI (blue), respectively. (e) HE staining and TUNEL staining of metastatic lymph nodes 24 h after in vivo PDT treatment with or without HAase treatment of their respective primary tumors. The right micrograph is a zoom-in image at the center of the lymph node with HAase-assisted PDT treatment.
Twelve days after 4T1 tumor cells inoculation at both rear foot pads, one side was injected with 3000 U HAase, while the other side was injected with saline as a control. Two hours after injection, NM-Ce6 was iv injected. The in vivo PDT was conducted 24 h post injection, by laser irradiation on the areas with SLNs. One day after the final treatment, the lymph nodes on both sides were taken out for H&E and TUNEL staining. In the center of the metastatic lymph node in the HAase treated group, a very large area of cell damage as well as abundant apoptotic cells could be observed in H&E and TUNEL stained images, respectively. In marked contrast, for the lymph nodes from the other side without HAase treatment, PDT treatment failed to induce significant cell damage or apoptosis (Figure
5e). Therefore, by using the foot pad lymphatic metastasis model, we demonstrate that the combination of HAase treatment and PDT could bring synergistic effects for elimination of metastatic SLNs, owing to the remarkably enhanced EPR effect and relieved hypoxic state in those SLNs. Such an effective treatment of metastatic SLNs would be helpful to inhibit further lymphatic tumor metastasis and prolong animal survival, as demonstrated in a number of our previous studies.56,58 6. Clinical Translational Tendency of HAase Combined with PDT. As for clinical translation purpose, the previous strategy of intratumoral injection of HAase might have be certain limitations.61 Instead of local injection, systemic 2518
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Figure 6. Enhanced EPR effect and relieved hypoxia state of tumor after intravenous injection of HAase. (a) Fluorescence imaging of 4T1 tumor bearing mice at different time points (6, 10, and 24 h) after intravenous injection of saline plus NM-Ce6 or HAase plus NM-Ce6. (b) Statistic data of mean fluorescence intensity on tumors for two groups at different time points. Student’s t test were used. (* indicates p < 0.05, and ** indicates p < 0.01) (c) Immunofluorescence imaging of tumor slices 24 h after injection. Blue, green, and red channels indicate DAPI, hypoxia region, and blood vessels with CD31 expression, respectively.
intravenous injection would be preferred owing to its noninvasiveness and high operability. To this end, HAase modified with N-hydroxylsuccinimide functionalized polyethylene glycol (Mw = 5000, NHS-PEG5000) was iv injected with NM-Ce6 simultaneously. The fluorescence imaging of 4T1 tumor bearing mice showed that the tumor fluorescence in HAase-PEG plus NM-Ce6 injection group was significantly higher than that in the saline plus NM-Ce6 injection group, suggesting the improved EPR effect of 4T1 tumors after systemic administration of PEGylated HAase (Figure 6a,b). Furthermore, the hypoxia states of tumors in both groups were also examined 24 h after iv injection of HAase-PEG. The green hypoxia-stained fluorescence in the tumor from HAase-PEG injected group appeared to be significantly weaker than that in the saline injected group, revealing that the inherent hypoxia state of the tumor was also greatly relieved owing to the systemic administration of PEGylated HAase (Figure 6c). However, the enhanced EPR effect in the tumor after systemic injection of PEGylated HAase appeared to be no better than that achieved after local injection of HAase, likely owing to the reduced effective dose of HAase reaching the tumor with iv injection. No significant increase in the blood vessel density was observed in the tumor after iv injection of HAase-PEG, which, however, resulted in a higher percentage of effective vascular area (Figure 6c), a possible reason for the obviously relieved hypoxia state in the tumor. Although being slightly less effective in our experiment with our homemade PEGylated HAase, systemic injection of HAase compared with local injection would have more significant value for future clinical translation. The several unfavorable
drawbacks of local injection, such as being invasive (with the risk to damage tumor boundary and trigger metastasis), uneven distribution of the administrated drug inside a large tumor, and the possibility to miss some small undetected tumors, could be avoided by systemic administration.62 Fortunately, PEGylated HAase with the optimized formulation, high enzyme activity, and great in vivo efficacy useful for systemic administration has been developed by Halozyme Therapeutics under the trade name of PEGPH20.32,46 It is thus hoped that the strategy proposed in this work may indeed bring new opportunities to improve PDT cancer treatment in the clinic. Conclusions. In summary, we have demonstrated a new approach to improve the nanomedicine-based photodynamic therapy in cancer treatment by modulating the tumor microenvironment via administration with HAase, which would bring the following benefits for PDT. First, both the densities of tumor blood vessels and percentages of dilated vessels are increased after HAase treatment, promoting the tumor perfusion and alleviating the hypoxia state of tumor. Second, the EPR effect of tumors for nanoparticles could be enhanced due to the decomposition of extracellular matrix, reduced IFP, and improved tumor perfusion. Lastly, HAase could migrate from primary tumors to their drainage SLNs, enhancing the EPR effect and relieving the hypoxic state in those lymph nodes with metastatic tumor cells. As a result, we achieved fantastic synergistic therapeutic effects by combining HAase administration with nanoparticle-based PDT in both the subcutaneous breast cancer model and the lymph node metastasis model. In addition, systemic injection of PEGylated HAase could also bring similar benefits as local intratumoral 2519
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injection, suggesting the potential clinical translation tendency of this strategy. Our work presents a novel strategy to enhance the in vivo PDT efficacy by altering the inherent tumor microenvironment in both primary tumors and their metastatic lymph nodes, potentially beneficial for broadening the clinical applications of PDT. Moreover, our results also suggest that tuning the abnormal characteristics of tumor microenvironment may be a useful approach to improve and optimize various types of nanomedicine-based cancer therapies, as well as other conventional therapeutic strategies currently applied in the clinic.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b00068. Experiment section and supplementary Figures S1−S4. (PDF) Video of saline treatment. (AVI) Video of HAase treatment. (AVI)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partially supported by the National Basic Research Programs of China (973 Program) (2012CB932600), the National Natural Science Foundation of China (51525203, 51132006), the Collaborative Innovation Center of Suzhou Nano Science and Technology, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.
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