Letter pubs.acs.org/NanoLett
Hyaluronidase Embedded in Nanocarrier PEG Shell for Enhanced Tumor Penetration and Highly Efficient Antitumor Efficacy Hao Zhou,† Zhiyuan Fan,† Junjie Deng,† Pelin K. Lemons,† Dimitrios C. Arhontoulis,‡ Wilbur B. Bowne,§ and Hao Cheng*,†,‡ †
Department of Materials Science and Engineering, and ‡School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania 19104, United States § Department of Surgery, Drexel University, Philadelphia, Pennsylvania 19102, United States S Supporting Information *
ABSTRACT: One of the major challenges in applying nanomedicines to cancer therapy is their low interstitial diffusion in solid tumors. Although the modification of nanocarrier surfaces with enzymes that degrade extracellular matrix is a promising strategy to improve nanocarrier diffusion in tumors, it remains challenging to apply this strategy in vivo via systematic administration of nanocarriers due to biological barriers, such as reduced blood circulation time of enzymemodified nanocarriers, loss of enzyme function in vivo, and life-threatening side effects. Here, we report the conjugation of recombinant human hyaluronidase PH20 (rHuPH20), which degrades hyaluronic acid, on the surfaces of poly(lactic-coglycolic acid)-b-polyethylene glycol (PLGA-PEG) nanoparticles followed by anchoring a relatively low density layer of PEG, which reduces the exposure of rHuPH20 for circumventing rHuPH20-mediated clearance. Despite the extremely short serum half-life of rHuPH20, our unique design maintains the function of rHuPH20 and avoids its effect on shortening nanocarrier blood circulation. We also show that rHuPH20 conjugated on nanoparticles is more efficient than free rHuPH20 in facilitating nanoparticle diffusion. The facile surface modification quadruples the accumulation of conventional PLGA-PEG nanoparticles in 4T1 syngeneic mouse breast tumors and enable their uniform tumor distribution. The rHuPH20-modified nanoparticles encapsulating doxorubicin efficiently inhibit the growth of aggressive 4T1 tumors under a low drug dose. Thus, our platform technology may be valuable to enhance the clinical efficacy of a broad range of drug nanocarriers. This study also provides a general strategy to modify nanoparticles with enzymes that otherwise may reduce nanoparticle circulation or lose function in the blood. KEYWORDS: Extracellular matrix, hyaluronan, appoptosis, heterogeneous, drug release
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acid (HA) for enhancing drug penetration in tumors.18,19 HA is a major component of ECM, and its accumulation contributes to the increase of interstitial fluid pressure in tumors, reducing therapeutic diffusion.20 High levels of tumor HA have been observed in many cancer patients.21−23 The interaction between HA and cancer cells is correlated to cancer metastasis and short overall survival of patients.22 In preclinical studies, a high dose of PEGPH20 has been used systemically to deplete tumor HA prior to the injection of therapeutics.21,24 Initial clinical studies with PEGPH20 have shown promising results but side effects, such as muscle spasm and thromboembolism, have been reported.18,19 We hypothesize that conjugated hyaluronidase on NPs surfaces enhances NP penetration in solid tumors, enables a high efficient utilization of the enzyme, and avoids unnecessary degradation of a large amount of HA as
anocarriers/nanoparticles (NPs) improve pharmacokinetics of encapsulated drugs and accumulate in tumors due to the leaky vasculature and impaired lymphatic drainage of tumor tissues,1−3 which make them a promising modality in cancer therapy. A few nanomedicines have been approved by U.S. Food and Drug Administration,4,5 and many more are under clinical trials.6,7 Despite the success, one major challenges in applying nanomedicines in cancer therapy is the low interstitial diffusion of NPs after entering perivascular areas in solid tumors.8,9 Released drugs only penetrate a few layers of cells and are inaccessible to hypoxic tumor cells that are usually resistant to chemo- and radiotherapies.10,11 Elevated density of cells and extracellular matrix (ECM), high interstitial pressure, and the heterogeneous vasculature in tumors are the major reasons for the ineffective NP diffusion.9 Strategies have been explored to enhance therapeutic diffusion in tumors, but many have limitations for potential clinical use.12−17 One promising strategy under clinical trials is to use PEGylated rHuPH20 (PEGPH20) that degrades hyaluronic © XXXX American Chemical Society
Received: February 25, 2016
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DOI: 10.1021/acs.nanolett.6b00820 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 1. Preparation and characterization of hyaluronidase-modified nanocarriers. (a) Schematic illustration of NP fabrication and penetration in tumors via the degradation of hyaluronic acid (HA). The NPs were fabricated by conjugating thiolated rHuPH20 on the first PEG layer followed by anchoring the second PEG layer. (b) A representative TEM image of HPEG-PH20-NPs (Scale bar: 100 nm). (c) Quantification of the effective number of conjugated rHuPH20 on NPs prepared with three conditions. The rHuPH20 activity was assessed by a microtiter-based assay quantifying HA degradation. Values indicate mean ± SD (n = 3). (d) NP diffusion in ECM-mimicking gels. (Scale bar: 200 μm). The gels composed of 6.5 mg/ mL of rat collagen I and 1 mg/mL of HA in capillary tubes. Ten microliters of 1 mg/mL of NPs (green) were added on the top of gels and incubated at 37 °C for 1.5 h before being imaged. The activity of free or conjugated rHuPH20 was 500 U/mL. (e) Normalized NP fluorescence with diffusion distance in gels. Images were analyzed via ImageJ. Diffusion coefficients were obtained by fitting the data to a one-dimension diffusion model in MATLAB. Black lines display theoretical intensity profiles for particles with diffusion coefficients of 1.66 × 10−7, 7.17 × 10−8, and 1.11 × 10−8 cm2· s−1.
and demonstrate that the conjugated rHuPH20 is more efficient than free rHuPH20 to enhance NP diffusion in matrix, does not alter the NP circulation time and maintains its enzyme activity in the blood because of the extra PEG layer, and significantly improves NP accumulation in 4T1 syngeneic mouse breast tumors due to the increased tumor penetration. As a result of these effects, the modified NPs exhibit an antitumor efficacy superior to unmodified NPs. Conventional PLGA-PEG-NP was selected for this study because it is a widely used drug nanocarrier in cancer research. Improving its antitumor efficacy through enhancing particle tumor penetration would validate our strategy and pave the way to apply the technology in other drug nanocarriers. Our study also provides a general strategy to decorate NP surfaces with enzymes that otherwise may reduce NP circulation or lose function in the blood. To fabricate PLGA-PEG-NPs for enzyme modification, methoxy PLGA20K-PEG5K was mixed with maleimide-terminated PLGA20K-PEG5K at a 4:1 ratio to form NPs via a
NPs only degrade matrix on their diffusion path while releasing therapeutics. The strategy of modifying NPs with ECM-degrading enzymes has been previously explored for enhancing NP diffusion in tumors using collagenase,25,26 bromelain,27 or hyaluronidase.28 However, these studies were performed either in vitro using tumor spheroids or in vivo via local injection of NPs into tumors. Although systematic administration of NPs via intravenous injection is highly clinically relevant in cancer therapy, ECM-degrading enzyme-modified NPs have not been successfully applied in animal tumor models using this administration route due to biological barriers, such as reduced NP blood circulation time, loss of enzyme function in vivo, and life-threatening side effects. Here, we report a unique strategy to overcome these problems by adding an extra polyethylene glycol (PEG) layer around both NPs and conjugated ECM-degrading enzymes. We show the modification of poly(lactic-co-glycolic acid)-b-polyethylene glycol NP (PLGA-PEG-NPs) surfaces with rHuPH20 B
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Figure 2. Analysis of HPEG-NP internalization by cancer cells with pericellular HA matrix. (a) Confocal microscopy images of 4T1 cells treated with 0.02 mg/mL of either HPEG-NPs or HPEG-PH20-NPs with enzyme activity of 10 U/mL for 0, 1, 2, and 4 h. HA, nuclei, and DiD-labeled NPs were shown in green, blue, and red, respectively. The signal included both internalized particles and particles bound on cell surfaces (Scale bar: 50 μm). (b) Quantitative analysis of NP and (c) HA fluorescence intensities per 4T1 cell. Values indicate mean ± SD (n = 10) **P < 0.01. The rHuPH20modified NPs showed an enhanced internalization by 4T1 cells after crossing the HA matrix at each time point. HA quantitative analysis showed a gradual degradation of HA around 4T1 cells by rHuPH20 on the NPs. (d) Quantitative analysis of NP and (e) HA fluorescence intensity in 4T1 cells, which were treated for 2 h with 0.02 mg/mL of HPEG-NPs supplemented with free rHuPH20 at 0, 10, 100, or 1000 U/mL or treated with HPEG-PH20-NPs with 10 U/mL of enzyme activity. HA staining showed increased HA degradation as the increase of free rHuPH20 concentration. Considering the results of HA degradation and the amount of NPs that penetrate the HA matrix, rHuPH20 on NPs was significantly more efficient than free rHuPH20 in facilitating NP penetration. Values indicate mean ± SD (n = 10). **P < 0.01.
reduces the rHuPH20 activity by 43 ± 19% (Supporting Information Figure S3). This result demonstrates that the PEG2K molecules, although not conjugated on rHuPH20, can reduce the access of HA to rHuPH20 from lateral directions. In other studies of NP surface modification, bioactive molecules are mostly on the outmost layer.30 Our strategy partially embeds rHuPH20 in the PEG shell. This unique structure likely decreases the exposure of rHuPH20 to opsonins in the blood and avoids rHuPH20-mediated clearance of NPs in circulation. Collagen is the most abundant ECM protein and forms the scaffold, while HA associates with collagen and fills up the space in between collagen fibers. To study the effect of rHuPH20 on NP diffusion, gels composed of 6.5 mg/mL of rat tail collagen I and 1 mg/mL of HA were developed to mimic tumor ECM (Figure 1d).31 This physiological concentration is comparable to previous collagen gel models.12,16 PLGA-PEG-NPs conjugated with methoxy PEG2K-SH alone without rHuPH20 (HPEG-NPs) barely diffused into the gels, likely because of the small pore size of gels. The degradation of HA by rHuPH20 enabled NP diffusion (Figure 1d). The diffusion profiles were fitted to a one-dimensional diffusion model following a reported method (Figure 1e).12 Free rHuPH20 increased the diffusion coefficient of NPs from 1.09 ± 0.51 × 10−8 to 7.33 ± 0.25 × 10−8 cm2·s−1, while NPs with the same amount of effective rHuPH20 conjugated on the surfaces showed a diffusion coefficient of 1.72 ± 0.33 × 10−7 cm2·s−1 (mean ± SD, n = 3). When NPs and free rHuPH20 are simply mixed together, the HA degradation and NP diffusion may not occur at the same areas, causing a relatively low efficiency of rHuPH20-enhanced diffusion. However, rHuPH20 conjugated
nanoprecipitation method.29 The rHuPH20-modified PLGAPEG-NPs (HPEG-PH20-NPs) were prepared by a sequential conjugation of thiolated rHuPH20 (110 000 U/mg), methoxy PEG2K-maleimide (PEG2K-MAL), and PEG2K-thiol (PEG2KSH) on PLGA-PEG-NPs (Figure 1a). The rationale of conjugating an extra layer of PEG after anchoring rHuPH20 on PLGA-PEG-NPs is to minimize the effect of rHuPH20 on the circulation half-life of NPs because rHuPH20 has a serum half-life shorter than 3 min24 and significantly reduces NP particle blood circulation (Supporting Information Figure S1). Transmission electron microscopy (TEM) image of HPEGPH20-NPs showed an average size of 50 nm (Figure 1b), while the size measured with DLS was approximately 90 nm (Supporting Information Figure S2a). After NP dialysis, the complete removal of unreacted rHuPH20 was confirmed with a size exclusion assay (Supporting Information Figure S2b). The activity of rHuPH20 was only detected in the eluent fraction of NPs, which was determined using fluorescently labeled HPEGPH20-NPs. The amount of rHuPH20 on NPs can be controlled by adjusting the reaction conditions (Figure 1c). Condition 2 was employed to prepare HPEG-PH20-NPs for the rest of studies, resulting in NPs with 500 U of effective rHuPH20 activity per mg of polymer. The effect of the extra PEG layer on the enzyme activity of surface conjugated rHuPH20 was also investigated. Interestingly, it was found that methoxy PEG2K-MAL directly conjugated on rHuPH20 barely reduces NP enzyme activity, indicating the density of PEG chains linked on rHuPH20 was not high enough to shield HA from accessing rHuPH20. Further conjugation of PEG2K-SH on the primary PEG layer C
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Figure 3. Cytotoxicity of DOX-encapsulated HPEG-NPs with and without rHuPH20 modification. (a) In vitro DOX release from DOX-HPEG-NPs and DOX-HPEG-PH20-NPs. Values indicate mean ± SD (n = 4). NPs at 1 mg/mL were shaken at 200 rpm at 37 °C in dialysis against PBS supplemented with 10% FBS, and the released DOX was measured via its fluorescence intensity. (b) Measurement of cell viability using an MTT assay. The 4T1 cells were incubated with either HPEG-NPs, HPEG-PH20-NPs, free DOX, DOX-HPEG-NPs, or DOX-HPEG-PH20-NPs in 96-well plate for 4 h. The DOX concentrations were 3 μg/mL for all the treatments containing DOX. The cells were then washed with PBS and cultured in fresh cell culture medium for another 20 h. Values indicate mean ± SD (n = 3). **P < 0.01. (c) Confocal microscopy images of 4T1 cells treated with NPs encapsulating 3 μg/mL DOX. After 4 h incubation, the coverslips were washed and fixed for confocal microscopy imaging (scale bar: 20 μm).
NPs need to pass through the pericellular HA matrix prior to being internalized by cells, our observations suggest that degrading HA on the diffusion path of NPs is more efficient in facilitating NP penetration than random HA degradation as used in the strategy of applying free PEGPH20.24 The data are consistent with the result of NP diffusion in ECM-mimicking gels. To further confirm that the increased NP internalization is primarily a result of the enhanced ability of NPs to penetrate the pericellular matrix, 4T1 cells were pretreated with 3000 U rHuPH20 and maintained in 1000 U rHuPH20 to exclude the HA layer. When the HA matrix layer was reduced and no longer was an obstacle for NPs, cells treated with HPEG-NPs and HPEG-PH20-NPs showed a similar fluorescence intensity of NPs (Supporting Information Figure S5a,b), indicating the HA matrix is truly a barrier for NPs to access 4T1 cells. To investigate the possibility of rHuPH20-mediated binding, cells were treated with NPs alone after the pretreatment without free rHuPH20. The amount of internalized HPEG-NPs increased after pretreatment but was lower than that of HPEG-PH20NPs and the condition with free rHuPH20. This is likely because of the recovery of HA within the treatment time (Supporting Information Figure S5b). This result cannot rule out the possible effect of rHuPH20-mediated binding on NP internalization but demonstrates that the enhanced penetration is a major reason for the increased NP internalization. To investigate the therapeutic potential of HPEG-PH20-NP as a drug carrier, doxorubicin (DOX) was encapsulated in NPs with a final loading yield of 3.5 ± 0.1% and 3.4 ± 0.2% for NPs with and without rHuPH20 modification, respectively. There was a 20% release of DOX in the first 1 h in phosphate-buffered saline (PBS) supplemented with 10% fetal bovine serum (FBS) at 37 °C, and 30% of DOX remained encapsulated after 3 days (Figure 3a). The modification of HPEG-NPs with rHuPH20 did not alter the release profile of DOX (Figure 3a). The
on HPEG-NP surfaces generates a high local concentration of rHuPH20 around NPs that degrades HA on their diffusion path. In addition to stromal HA, many cancer cells have a pericellular HA matrix, preventing the access by NPs. Mouse 4T1 breast cancer cells synthesize HA and form a pericellular HA matrix.32 Therefore, 4T1 cells were selected to study the effect of conjugated rHuPH20 on cell internalization of NPs. The existence of HA matrix around 4T1 cells and their degradation by rHuPH20 were confirmed by HA staining (Supporting Information Figure S4a,b) and a red blood cell exclusion assay (Supporting Information Figure S4c,d). The internalization of 1,1-dioctadecyl-3,3,3,3-tetramethylindo-carbocyanine perchlorate (DiD)-labeled NPs was studied using confocal microscopy and analyzed with ImageJ. The signals were from internalized NPs and might also include cell membrane-bound NPs. In the study, 4T1 cells were treated with 0.02 mg/mL of either HPEG-PH20-NPs with 10 U/mL of rHuPH20 activity or HPEG-NPs for 1, 2, or 4 h. It was demonstrated that rHuPH20 modification enhanced NP internalization by 4T1 cells (Figure 2a,b). Although HPEGPH20-NPs degraded a significant amount of pericellular HA (Figure 2a,c), a complete removal of HA layer is unnecessary for the enhanced internalization as the enhancement appeared at early times when the majority of HA still remained. When HPEG-PH20-NPs with 10 U activity were compared to HPEGNPs mixed with 10, 100, or 1000 U of free rHuPH20, HPEGPH20-NPs again showed their superior efficiency. The internalization of HPEG-PH20-NPs by 4T1 cells was over 2 times higher than HPEG-NPs mixed with 10 U of free rHuPH20, the same amount of enzymatic activity (Figure 2d), while the HA degradation was almost equivalent in both cases (Figure 2e). More interestingly, HPEG-NPs mixed with 100 U of free rHuPH20 degraded more HA but still showed less internalization than HPEG-PH20-NPs (Figure 2d,e). Because D
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Figure 4. Biodistribution and tumor accumulation of HPEG-NPs with and without rHuPH20 modification in 4T1 syngeneic mouse tumor model. (a) In vivo circulation of DiD-labeled HPEG-NPs and HPEG-PH20-NPs measured by fluorescence intensity of NPs and by the enzyme activity of free rHuPH20 and HPEG-PH20-NPs in the plasma. Value indicates mean ± SD (n = 3−6). (b) Calculated circulation half-lives of HPEG-NPs, HPEG-PH20-NPs, and enzyme activity of free rHuPH20 and HPEG-PH20-NPs from one-compartment model. Values indicate mean ± SD (n = 3− 6). (c) IVIS fluorescence image of mice at 1, 3, 6, and 24 h post injection of either saline (i), HPEG-PH20-NPs (ii), HPEG-NPs (iii) or PLGA-PEGNPs (iv). (d) IVIS fluorescence image of tissues 24 h postinjection. All NPs were labeled with DiD. (e) Quantification of NP biodistribution in mice by measuring the fluorescence intensity of homogenized tissues that were collected at 24 h post NP injection. Relative fluorescence intensities per gram of tumor, brain, heart, lung, liver, spleen, and kidney were evaluated. The rHuPH20-modified NPs showed a significantly higher accumulation in 4T1 tumors. Values indicate mean ± SD (n = 3). **P < 0.01.
dependent clearance of rHuPH20.24 To confirm the activity of rHuPH20 on NPs in the blood, the circulation half-life of rHuPH20 on NPs was measured based on their activity and determined to be 5.1 ± 1.1 h (Figure 4a,b). BALB/c mice bearing 4T1 tumors were used to study the biodistribution of DiD-labeled NPs. The prolonged NP circulation time results in higher NP accumulation in tumors (Figure 4c,d). Quantitative analysis was performed by measuring the DiD signal of homogenized tissues (Figure 4e). It is found that the extended circulation of NPs improved their accumulation in tumors 1-fold at 24 h post tail vein injection of NPs. HPEG-NPs showed higher signals in liver and spleen than the conventional PLGA-PEG-NPs without the second PEG layer (Figure 4d,e). This may be because of the fast capture of PLGA-PEG-NPs by phagocytic cells in these tissues, and some NPs had already been degraded by the time of sample collection. It has been shown that enhanced therapeutic penetration in tumors increases therapeutic accumulation in tumors.13 As expected, modification of HPEG-NPs with rHuPH20 further improved NP accumulation in tumors by another 100%. Because the modification does not alter NP circulation time, the rHuPH20-mediated matrix penetration increased NP accumulation in tumors. HPEGPH20-NPs showed slightly higher NP signals than HPEG-NPs in liver and spleen of mice, possibly due to the same effect of enhanced matrix penetration. Compared to the conventional PLGA-PEG-NPs, the modification strategy for enhanced penetration increased NP accumulation in tumors by approximately 300%. The triple negative 4T1 syngeneic mouse tumor closely mimics human breast cancer,33 which is ideal for evaluating the antitumor efficacy of NPs. The tumor-bearing mice were not treated until day 9 post-orthotopic injection of 4T1 cells. By
therapeutic potential of DOX-encapsulated NPs was evaluated in vitro using the MTT assay. In the test, 4T1 cells were incubated with either NPs alone, NPs encapsulating 3 μg/mL of DOX, or the same amount of free DOX for 4 h and were cultured in regular medium for another 20 h before a viability test. The viability of cells treated with DOX-encapsulated HPEG-PH20-NPs (DOX-HPEG-PH20-NPs) was 47%, which is significantly lower than cells treated with DOX-encapsulated HPEG-NPs (DOX-HPEG-NPs) or free DOX, while the NPs alone showed no cytotoxicity (Figure 3b). In confocal images, cells that had been treated with DOX-encapsulated NPs for 4 h showed a DOX signal in nuclei (Figure 3c). There was a higher DOX accumulation in DOX-HPEG-PH20-NP-treated 4T1 cells than in DOX-HPEG-NP-treated cells, supporting the result from viability test. One technical challenge to apply nanocarriers with surfaceconjugated enzymes in vivo is the maintenance of nanocarrier blood circulation and enzyme function. We have minimized the effect of conjugated rHuPH20 on nanocarrier circulation by anchoring rHuPH20 in between two PEG layers. The measured half-lives of HPEG-NPs and HPEG-PH20-NPs were 9.3 ± 0.65 and 8.7 ± 0.39 h, respectively (Figure 4a,b). Without the extra PEG layer, the rHuPH20 on NP surfaces would significantly reduce NP blood circulation (Supporting Information Figure S1a,b). Interestingly, the circulation half-life of the conventional PLGA-PEG-NPs made of 100% of methoxy PLGA20K-PEG5K was approximately 3 h, significantly lower than that of HPEGNPs. This discovery will be further studied in the future. The circulation half-life of rHuPH20 was detected to be 1.4 min in our study (Figure 4a,b). Although this data may not be accurate because of the short interval between blood sample collections, it is consistent with the previous finding that the half-life of rHuPH20 is less than 3 min because of the high-mannoseE
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Figure 5. Antitumor efficacy of HPEG-PH20-NPs in 4T1 tumor-bearing BALB/c mice. (a) In vivo tumor growth inhibition curves for 4T1 tumorbearing mice that were treated with either saline, free DOX, DOX-HPEG-NPs or DOX-HPEG-PH20-NPs. The dose of DOX was 2 mg/kg for free DOX and DOX-encapsulated NPs. Values indicate mean ± SD (n = 6). **P < 0.01. Black arrows indicate the time of injection. (b) Survival rate plots show the percentage of animals remained alive in the study. Mice were sacrificed and were no longer counted for survival rate when their tumor size exceeded 2000 mm3. **P < 0.01. (c) TUNEL staining of sectioned tumor tissues that were collected after the completion of all doses for the groups treated with saline, free DOX, DOX-HPEG-NPs, and DOX-HPEG-PH20-NPs. Green, TUNEL; blue, nuclei. Scale bar: 100 μm. (d) Staining of sectioned tumor trusses that were collected 24 h post the administration of saline or NPs. The mice were iv injected with saline or DiD-labeled NPs on day 9 post 4T1 cell inoculation. Left column showed the staining of CD31 (green, representing blood vessels) and the distribution of NPs (red). The middle column showed the HA staining and the right column showed the α-SMA staining for the four study groups. Scale bar: 50 μm (left); 200 μm (middle); 100 μm (right).
that time, the average tumor sizes had reached 180 mm3, which is larger than the starting tumor size of 50 or 100 mm3 in many in vivo efficacy studies of nanomedicines.6 Tumors with larger sizes at a later stage have a more mature ECM to better mimic the condition in cancer treatment. The 4T1 tumor-bearing mice were treated by tail vein injection of either DOX-HPEGNPs, DOX-HPEG-PH20-NPs, free DOX, or saline. The dose of DOX was 2 mg/kg, while the enzyme activity of DOX-HPEGPH20-NPs was 500 U/mouse. The antitumor efficacy of DOXHPEG-NPs with free enzyme can be a control but was not studied here because rHuPH20 has an extremely short half-life in the blood (Figure 4a,b), not suitable for intravenous injection.24 PEGPH20 combined with chemotherapy drugs is under clinical trials.19 When it was used in animal tumor models, the enzyme activity was approximately 10 times of that in our study.24 The 4T1 tumor-bearing mice were treated with five doses until the control group of saline-treated mice reached the criteria for euthanization. All the mice in the DOX-treated groups showed slower tumor growth and survived longer than the control group of mice (Figure 5a,b). DOX-HPEG-NPs showed slightly better antitumor efficacy than free DOX in terms of reducing primary tumor growth. DOX-HPEG-PH20NPs exhibited an antitumor efficacy superior to free DOX and NPs without rHuPH20 modification (P < 0.01). The treatment efficiently inhibited the growth of aggressive 4T1 tumors in spite of a low DOX dose. The percent increased life span (ILS) was calculated for each group (Supporting Information Figure S6a). Free DOX and DOX-HPEG-NPs-treated mice showed ILSs of 10% and 15%,
while mice treated with DOX-HPEG-PH20-NPs showed a significant higher ILS of 35% even though the treatment was stopped on day 20. The mouse weight did not show significant difference between each study groups (Supporting Information Figure S6b), indicating the treatment of DOX-HPEG-PH20NPs is well tolerated in mice. Obviously, the increased NP accumulation in tumors is a major reason for the antitumor efficacy of DOX-HPEG-PH20NPs. To further understand the antitumor mechanism of rHuPH20-modified NPs, tumor cell apoptosis, NP spatial distribution, and variation of tumor HA and activity of tumorassociated fibroblasts (TAFs) before and after treatment were analyzed. Apoptotic cells in the dissected 4T1 tumor tissues were stained with TUNEL. The representative confocal microscopy images showed that DOX-HPEG-PH20-NPs were more efficacious than other treatments to induce tumor cell apoptosis (Figure 5c). Furthermore, the TUNEL staining was more uniformly distributed in tumors treated with rHuPH20modified NPs than those treated with rHuPH20-free NPs. To demonstrate the spatially uniform distribution of apoptotic tumor cells was from the enhanced NP penetration, tumors were harvested 24 h after systemic administration of DiDlabeled NPs. The sectioned tumor tissues were stained with CD31, marking blood vessels (Figure 5d left). There was barely detectable NP signal (red) in tumors treated with PLGA-PEGNPs due to the short circulation time of the NPs, consistent with the biodistribution study. With a longer circulation time, HPEG-NPs were able to accumulate more into tumors. Significantly higher and better-distributed NP signals around blood vessels were observed in tumors treated with HPEGF
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SH) and Methoxy-poly(ethylene glycol)-maleimide (Mw ∼ 2000) (PEG-MAL) were purchased from Laysan Bio Inc. Mouse breast cancer cell line, 4T1, was purchased from ATCC Inc. Horseradish peroxidase conjugated streptavidin (HRPStreptavidin), o-phenylenediamine (OPD), 2-iminothiolane· HCl (Traut’s Reagent), N-hydroxysulfosuccinimide (SulfoNHS), 1-ethyl-3-(3-dimethylamin-opropyl) carbidodiimide (EDC), and (biotinyl)hydrazide (Biotin hydrazide) were purchased from Thermo Scientific. DiD oil (DilC18(5) oil) and Alexa Fluor 488-conjugated streptavidin were products of Life Technologies. Mouse monoclonal anti-α-SMA antibody and goat antimouse IgG H&L (Alexa Fluor 488) were purchased from Abcam, rat anti-Mouse CD31 antibody and Alexa Fluor 488-Goat antirat IgG were purchased from BD Pharmingen and Jackson ImmunoResearch Inc. respectively, while biotinylated HA binding Protein was obtained from EMD Millipore (CalBioChem). Additional salts, solvents and buffers were purchased from Fisher Scientific. Fabrication of HPEG-PH20-NPs. PLGA-PEG-NPs were prepared using a nanoprecipitation method.29 Briefly, methoxy PLGA20k-PEG5K and PLGA20k-PEG5K-maleimide (PLGA20kPEG5K-MAL) (4:1 w/w or an indicated ratio) were dissolved in acetonitrile at a total polymer concentration of 5 mg/mL. To label NPs, a fluorescent dye, 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocy anine perchlorate (DiD), was also dissolved in acetonitrile and added at a weight ratio of 0.2% to polymers. The acetonitrile solution of polymers was added dropwise into PBS (2:5 v/v) and was kept stirring for 3 h before being kept under home vacuum overnight. The rHuPH20 was first thiolated via Traut’s reaction. Five-fold of Traut’s reagent was added into 1 mg/mL of rHuPH20 solution in PBS and reacted for 1 h at room temperature under stirring. After the reaction, the thiolated rHuPH20 was purified using a desalting column (Thermo Scientific.) and then added into the PBS solution of PLGA-PEG-NPs at 100 μg/mL. After 2 h under stirring, PEG2k-maleimide (PEG2k-MAL) and PEG2k-thiol (PEG2k-SH) were subsequently added to the NP solution to quench the unreacted thiol on rHuPH20 and maleimide on NP surfaces, which added a second PEG layer on NPs with the conjugated rHuPH20 in the outer layer. The final concentrations of PEG2kMAL and PEG2k-SH were 0.1 and 2 mg/mL respectively. In the control study, PEG100-SH was applied to replace PEG2k-SH at a concentration of 0.2 mg/mL. The obtained HPEG-PH20-NPs were purified via dialysis against saline overnight through 50 nm membranes at 4 °C. NPs were concentrated through centrifugal filters (Amicon Ultracel 30K) and passed through a 0.2 μm syringe filter before further use. DiD-labeled PLGAPEG-NPs with the second PEG layer alone (HPEG-NPs) were prepared using the same method as the fabrication of DiDlabeled HPEG-PH20-NPs but without enzyme modification. NP Diffusion in ECM-Mimicking Gels. ECM-mimicking gels composed of collagen and HA were prepared by mixing the following components in order on ice: 48 μL of 10× PBS, 12.8 μL of 1N NaOH, 24.3 μL of DI water, 160 μL of HA at 5 mg/ mL (MW = 1.5−2.0 × 106) in 2× PBS and 555 μL of rat tail collagen type I (Corning, Bedford, MA) at 9.37 mg/mL. The final concentration of collagen was 6.5 mg/mL and HA was 1 mg/mL. The mixture was thoroughly vortexed and vacuumed on ice to remove bubbles before adding 60 μL of solution into each capillary tube (0.4 × 4.00 mm ID, Vitrocom, Mountain Lakes, NJ), the tubes were then incubated overnight at 37 °C. In the diffusion tests, 10 μL of equalized 1 mg/mL of DiDlabeled HPEG-NP, HPEG-NP + 500 U/mL of free rHuPH20,
PH20-NPs, which attributes to the pattern of apoptotic cells in tumors. HA staining of tumors, which had been treated for 24 h, did not show a significant difference in four treatments (Figure 5d middle) as evaluated by the quantitative analysis of HA staining of three tumors per study groups (Supporting Information Figure S7). This result indicates the majority of tumor HA remained intact under the treatment of HPEG-PH20-NPs because of the efficient utilization of the conjugated rHuP20 by the NPs, which only degrade HA on their diffusion path. The fast synthesis of new HA that compensated for HA degradation likely also attributes to the observation. Together with the data of NP distribution in tumors, we demonstrated that NPs with conjugated rHuPH20 is advantageous in term of minimizing unnecessary HA degradation. Similar results were obtained in a short treatment of 2h (Supporting Information Figure S8). TAFs play an important role in synthesizing and remodeling tumor ECM including HA.34 Reduction of TAF activity by NPcarried drug has been shown to improve NP penetration in tumors because of the decreased ECM density.35 To test if the enhanced HPEG-PH20-NP penetration in 4T1 tumors is due to a reduced TAF activity, a TAF activity marker, α-smooth muscle actin (α-SMA) of sectioned tumor tissues was stained (Figure 5d right) and quantified with ImageJ (Supporting Information Figure S9). There is no significant difference in αSMA expression among the four groups, demonstrating that the penetration of HPEG-PH20-NPs is not due to reduced TAF activity by the NPs. In summary, this study demonstrates that engineering NP surfaces with rHuPH20 enhances nanocarrier diffusion in matrix and penetration in tumors and improves the antitumor efficacy of nanomedicines significantly. Our unique doublePEG-layer strategy avoids the potential negative effect of rHuPH20 on NP blood circulation and maintains enzyme function in vivo. This approach is expected to have broad applications in NP surface modification. In current clinical trials, PEGPH20 is administered 1 day before applying anticancer agents to ensure the depletion of tumor HA.18,19 Hyaluronidase conjugated on NP surfaces is designed to degrade HA only on the diffusion paths of NPs. This characteristic efficiently utilizes hyaluronidase and may reduce potential side effects by avoiding unnecessary degradation of HA. Our strategy requires one injection of each dose, which is different from the current clinical strategy and therefore is expected to improve patient compliance. Because of the rapid degradation and synthesis of HA in humans, the temporary degradation of tumor HA is not expected to promote cancer metastasis, especially when the degradation is minor and results in direct exposure of cancer cells to therapeutics. Our technology is highly clinically relevant. It is promising to improve the efficacy of nanomedicines in clinical trials and enable the clinical applications of nanocarriers that were previously considered impractical due to rapid NP clearance in the blood or inefficient diffusion in solid tumors. Experimental Details. Materials. Recombinant human hyaluronidase PH20 in pH 6.5, 10 mM sodium phosphate, 150 mM NaCl buffer was obtained from Halozyme, Inc. Doxorubicin hydrochloride salt (DOX) was purchased from BioTang, Inc. Methoxy poly(ethylene glycol)-b-poly(lactic-coglycolic acid) (Mw ∼ 5000:20000) (PLGA-PEG) and poly(lactic-co-glycolic)-b-poly(ethylene glycol)-maleimide (Mw ∼ 20000−5000) (PLGA-PEG-MAL) were purchased from Akina Inc. Methoxy-poly(ethylene-glycol)-thiol (Mw ∼ 2000) (PEGG
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vivo blood circulation half-lives were calculated based on onecompartment model of pharmacokinetics via PKSolver. To quantify the enzyme activity half-life of free rHuPH20 and rHuPH20 on NPs, EDTA instead of heparin was used during blood collection. Free rHuPH20 was iv injected at 100 μL of 10 000 U/mL in saline. The remaining enzyme activity was measured using the microtiter-based assay described in the section of enzyme activity assay in the Supporting Information. All animal procedures were conducted according to the protocols of the Committee on Animal Care of Drexel in compliance with NIH guidelines. Mouse 4T1 Breast Cancer Model. Female BALB/c mice at 6-weeks old were ordered from Charles River Laboratory. After one-week, 50 μL of 1 × 107 4T1 cells/mL in PBS was orthotopically injected into the mammary fat pad of the mice on the right flank. Tumor size was calculated by V = 0.5 × width2 × length. Biodistribution Study. When the tumor size reached 200− 350 mm3, the mice were randomly divided into 4 groups and iv injected with either 150 μL at 10 mg/mL of DiD-labeled HPEG-PH20-NPs, HPEG-NPs, conventional PLGA-PEG-NPs made of 100% of methoxy PLGA20K-PEG5K, or saline. Mice were imaged at 1, 3, 6, and 24 h postinjection using IVIS Lumina XR. After 24 h, mice were sacrificed and their brains, lungs, hearts, livers, spleens, kidneys as well as tumors were collected and imaged with IVIS Lumina XR (605 nm excitation and Cy5.5 emission, 1s exposure). To quantify the NPs in each tissue, all the tissues were weighted and a fraction of the tissues were homogenized. The fluorescence intensities of homogenized tissue solution were detected via TECAN. Antitumor Efficacy and Survival Rate Study. The average tumor size reached 180 mm3 on day 9 post inoculation of 4T1 cells. The tumor-bearing mice were randomly divided into four groups. Each group was treated by tail vein injection of either DOX-HPEG-NPs, DOX-HPEG-PH20-NPs, free DOX, or saline on day 9, 11, 14, 17, and 20 post-4T1 cell inoculation. The dose of DOX was 2 mg/kg, whereas the enzyme activity of DOX-HPEG-PH20-NPs was 500 U/mouse. Tumor sizes and mouse body weight were monitored throughout the whole experiment. Mice were sacrificed when their tumor size reached 2000 mm3. The survival rate was studied based on the number of mice in each group over time. Median survival time (MST, day) was defined as the time when half of the mice died. The percent increased life span (ILS, %) was calculated using the following equation: ILS (%) = [(MST of treated group/MST of control group) −1] × 100. Apoptosis, CD31, HA, and α-SMA Staining of Sectioned Tumor Tissues. To study tumor cell apoptosis, one mouse from each study group of antitumor efficacy was sacrificed 2 days after the last dosing (day 22 post 4T1 cell inoculation). Their tumors were collected and frozen at −80 °C in M-1 Embedding Matrix (Thermo Scientific). Tumors were sectioned in a thickness of 10 μm for TUNEL staining (Life Technology) following the manufacturer’s instructions. To study NP spatial distribution in tumors, tumor HA expression, and the activity of TAFs after treatment, tumors collected from the biodistribution study were frozen at −80 °C in M-1 Embedding Matrix and were sectioned at 10 μm thick. The sectioned tumor specimens were fixed in acetone at −20 °C for 5 min and air-dried before being washed with PBS for three times. The specimens were blocked in 3% BSA in PBS solution for 1 h at room temperature. Rat anti-Mouse CD31 antibody for CD31 staining was diluted at 1:100, HA binding
and 1 mg/mL of HPEG-PH20-NP solution was slowly added on the surface of the ECM mimicking gels. The tube was sealed and kept at 37 °C for 1.5 h then imaged using a confocal laser scanning microscope. Image analysis was performed using ImageJ. Diffusion profiles of relative intensity (C) and the diffusion distance (x) for the NPs were fitted to the following one-dimensional diffusion model to obtain the diffusion coefficient D in the ECM mimicking gel ⎛ x ⎞ ⎟+B C(x , t ) = A × erfc⎜ ⎝ 2tD ⎠
where erfc is the complementary error function and A, B are the constant for the function. The nonlinear curve fitting was performed by using fminsearch in MATLAB. Encapsulation of Doxorubicin in NPs. To fabricate NPs encapsulating DOX, DOX hydrochloride salt was first dissolved in acetonitrile/methanol (9:1 v/v) at a concentration of 10 mg/ mL and reacted with 5-fold triethylamine (TEA) at roomtemperature overnight. The DOX solution was then added into the polymer solution with a ratio of 10% (wt %). NPs were then fabricated using the above-described method by adding polymer solution into PBS dropwise. The NPs were then conjugated with thiolated-rHuPH20 and/or second PEG layer. The amount of encapsulated DOX was quantified by measuring the fluorescence intensity of DOX (excitation at 480 nm and emission at 595 nm). Twenty microliters of DOX-encapsulated NP sample was dissolved in 180 μL of DMSO, and the fluorescence intensity was read with a microplate reader (TECAN Infinite M200). Encapsulation yield was calculated through the following equation Loading yield =
Mass of DOX encapsulated in NP Mass of polymer in NP
MTT Assay for Cell Viability. The 4T1 cells were cultured with RPMI-1640 medium w/o Phenol Red in a 96-well plate 1 day before the MTT assay. After dilution, 200 μL of samples was added into each well at a DOX concentration of 3 μg/mL. Free DOX stock solution was prepared by dissolving 1 mg/mL of DOX in water and diluted in saline. The 4T1 cells were treated with either HPEG-PH20-NPs or HPEG-NPs with or without encapsulated DOX for 4 h. Then, the culture medium containing NP samples or free DOX were removed, and the 4T1 cells were washed 3 times with PBS before being cultured in fresh medium for another 20 h. At the end of the incubation, 20 μL at 5 mg/mL of Thiazolyl Blue Tetrazolium Bromide (MTT) in PBS was added to 100 μL medium per well. After incubation for 4 h, the medium was carefully removed and 200 μL of DMSO was added to each well. The absorbance at 570 nm deducting that at 630 nm was measured for each well after 5 min incubation and 3 min shaking. Relative cell viability was obtained by comparing to the untreated cells. All measurements were done in triplicate. Circulation Half-Life Study. DiD-encapsulated NPs at 10 mg/mL were systematically administered through tail vein injection. Each female BALB/c mouse received 100 μL of NP solution. A small volume of 15 μL of blood was collected at 2 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h post iv injection. The blood was diluted in 200 μL of PBS containing 16 U/mL heparin as an anticoagulant. Blood cells were removed by spinning at 300 g for 5 min, and 180 μL of the supernatant was used for testing fluorescence intensity with TECAN (Excitation at 600 nm and Emission at 665 nm). In H
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(4) Zhang, L.; Gu, F. X.; Chan, J. M.; Wang, A. Z.; Langer, R. S.; Farokhzad, O. C. Clin. Pharmacol. Ther. 2008, 83, 761−769. (5) O’Brien, M. E.; Wigler, N.; Inbar, M.; Rosso, R.; Grischke, E.; Santoro, A.; Catane, R.; Kieback, D. G.; Tomczak, P.; Ackland, S. P.; Orlandi, F.; Mellars, L.; Alland, L.; Tendler, C.; Group, C. B. C. S. Ann. Oncol 2004, 15, 440−449. (6) Hrkach, J.; Von Hoff, D.; Ali, M. M.; Andrianova, E.; Auer, J.; Campbell, T.; De Witt, D.; Figa, M.; Figueiredo, M.; Horhota, A.; Low, S.; McDonnell, K.; Peeke, E.; Retnarajan, B.; Sabnis, A.; Schnipper, E.; Song, J. J.; Song, Y. H.; Summa, J.; Tompsett, D.; Troiano, G.; Van Geen Hoven, T.; Wright, J.; LoRusso, P.; Kantoff, P. W.; Bander, N. H.; Sweeney, C.; Farokhzad, O. C.; Langer, R.; Zale, S. Sci. Transl. Med. 2012, 4, 128ra39. (7) Keefe, S. M.; Heitjan, D.; Hennessey, M.; Robinson, J.; Mykulowicz, K.; Marshall, A.; Gunnarsson, O.; Mamtani, R.; Vaughn, D. J.; Hoffman-Censits, J. H.; Nathanson, K. L.; Lal, P.; Pryma, D. A.; Eliasof, S.; Garmey, E. G.; Cohen, R. B.; Haas, N. B. J. Clin. Oncol. 2014, 32, 1. (8) Kratz, F.; Warnecke, A. J. Controlled Release 2012, 164, 221−235. (9) Chauhan, V. P.; Stylianopoulos, T.; Boucher, Y.; Jain, R. K. Annu. Rev. Chem. Biomol. Eng. 2011, 2, 281−298. (10) Davies, C de L.; Berk, D. A.; Pluen, A.; Jain, R. K. Br. J. Cancer 2002, 86, 1639−1644. (11) Minchinton, A. I.; Tannock, I. F. Nat. Rev. Cancer 2006, 6, 583− 592. (12) Wong, C.; Stylianopoulos, T.; Cui, J.; Martin, J.; Chauhan, V. P.; Jiang, W.; Popovic, Z.; Jain, R. K.; Bawendi, M. G.; Fukumura, D. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2426−2431. (13) Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Greenwald, D. R.; Ruoslahti, E. Science 2010, 328, 1031− 1035. (14) Eikenes, L.; Bruland, O. S.; Brekken, C.; Davies Cde, L. Cancer Res. 2004, 64, 4768−4773. (15) Diop-Frimpong, B.; Chauhan, V. P.; Krane, S.; Boucher, Y.; Jain, R. K. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2909−2914. (16) Tong, R.; Chiang, H. H.; Kohane, D. S. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 19048−19053. (17) Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Nat. Nanotechnol. 2011, 6, 815−823. (18) Hingorani, S. R.; Harris, W. P.; Beck, J. T.; Berdov, B. A.; Wagner, S. A.; Pshevlotsky, E. M.; Tjulandin, S.; Gladkov, O.; Holcombe, R. F.; Jiang, P.; Devoe, C. E. Clin. Cancer Res. 2016, 33, 1. (19) Hingorani, S. R.; Harris, W. P.; Hendifar, A. E.; Bullock, A. J.; Wu, X. H. W.; Huang, Y.; Jiang, P. J. Clin. Oncol. 2015, 33, 1. (20) Whatcott, C. J.; Han, H.; Posner, R. G.; Hostetter, G.; Von Hoff, D. D. Cancer Discovery 2011, 1, 291−296. (21) Provenzano, P. P.; Cuevas, C.; Chang, A. E.; Goel, V. K.; Von Hoff, D. D.; Hingorani, S. R. Cancer Cell 2012, 21, 418−429. (22) Auvinen, P.; Tammi, R.; Kosma, V. M.; Sironen, R.; Soini, Y.; Mannermaa, A.; Tumelius, R.; Uljas, E.; Tammi, M. Int. J. Cancer 2013, 132, 531−539. (23) Aaltomaa, S.; Lipponen, P.; Tammi, R.; Tammi, M.; Viitanen, J.; Kankkunen, J.; Kosma, V. Urol. Int. 2002, 69, 266−272. (24) Thompson, C. B.; Shepard, H. M.; O’Connor, P. M.; Kadhim, S.; Jiang, P.; Osgood, R. J.; Bookbinder, L. H.; Li, X.; Sugarman, B. J.; Connor, R. J.; Nadjsombati, S.; Frost, G. I. Mol. Cancer Ther. 2010, 9, 3052−3064. (25) Kuhn, S. J.; Finch, S. K.; Hallahan, D. E.; Giorgio, T. D. Nano Lett. 2006, 6, 306−312. (26) Goodman, T. T.; Olive, P. L.; Pun, S. H. Int. J. Nanomed. 2007, 2, 265−274. (27) Parodi, A.; Haddix, S. G.; Taghipour, N.; Scaria, S.; Taraballi, F.; Cevenini, A.; Yazdi, I. K.; Corbo, C.; Palomba, R.; Khaled, S. Z.; Martinez, J. O.; Brown, B. S.; Isenhart, L.; Tasciotti, E. ACS Nano 2014, 8, 9874−9883. (28) Scodeller, P.; Catalano, P. N.; Salguero, N.; Duran, H.; Wolosiuk, A.; Soler-Illia, G. Nanoscale 2013, 5, 9690−9698.
protein (HABP) was diluted at 1:100 for HA staining, while anti-α SMA antibody (1A4) for α-SMA staining was diluted at 1:200. After incubation for 3 h at room temperature, the slides were washed with PBS for three times. The three kinds of samples were incubated with Alexa Fluor 488-Goat antirat IgG, Alexa Fluor 488-conjugated streptavidin, and Alexa Fluor 488Goat antimouse IgG respectively for 2 h at room temperature before washing and mounting. Fluorescence images were taken via confocal laser scanning microscopy. Statistical Analysis. Data are presented as mean ± SD. Statistical differences among experimental groups were analyzed using one-way analysis of variance (ANOVA) followed by two-tailed Student’s t test; P < 0.05 was considered statistically significant.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b00820. Additional methods. rHuPH20 on NP surface reduces the circulation of NPs in the blood (Figure S1). Characterization of rHuPH20-modified HPEG-NPs (Figure S2). Quantification of the effective enzyme activity of rHuPH20 on NPs before and after sequential conjugation of PEG2K-MAL and PEG2K-SH (Figure S3). Pericellular HA matrix around 4T1 cells and their depletion by rHuPH20 (Figure S4). The rHuPH20enhanced NP penetration through the pericellular HA matrix of 4T1 cells increases NP internalization by the cells (Figure S5). In vivo characterization of HPEGPH20-NPs (Figure S6). Characterization of tumor HA expression (2, 24 h) and the activity of actitumorassociated fibroblasts (24 h) post treatment with NPs and controls (Figure S7−S9). (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare the following competing financial interest(s): Hao Cheng and Hao Zhou are inventors of a patent filed by Drexel University. The patent covers the technology reported in this paper.
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ACKNOWLEDGMENTS This work was supported by a faculty startup fund from Drexel University to H.C. and a seed funding from the Clinical & Translational Research Institute (CTRI) to W.B.B. and H.C. We thank Halozyme Therapeutics for providing rHuPH20 and Mr. David Kang and Dr. Tzung Yang for the helpful discussion about hyaluronidase. We also thank Dr. Yichao Lu for his help on analyzing the data of nanoparticle diffusion in matrix and Chiemela Nwaobasi for training H.Z. to use Cryostat.
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REFERENCES
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