A Specific Drug Delivery System for Targeted Accumulation and

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A Specific Drug Delivery System for Targeted Accumulation and Tissue Penetration in Prostate Tumor Based on Microbially Synthesized PHBHHx Biopolyester and iRGD Peptide Fused PhaP Fan Fan, Xingjuan Wu, Jiping Zhao, Ganqiao Ran, Sen Shang, Mingchuan Li, and Xiaoyun Lu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00524 • Publication Date (Web): 23 Nov 2018 Downloaded from http://pubs.acs.org on November 24, 2018

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ACS Applied Bio Materials

A Specific Drug Delivery System for Targeted Accumulation and Tissue Penetration in Prostate Tumor Based on Microbially Synthesized PHBHHx Biopolyester and iRGD Peptide Fused PhaP Fan Fan,† Xingjuan Wu,† Jiping Zhao,† Ganqiao Ran,† Sen Shang,† Mingchuan Li,‡ and Xiaoyun Lu*† †

Department of Biological Science and Bioengineering, Key Laboratory of Biomedical

Information Engineering of the Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, Shaanxi, P. R. China ‡

Molecular Biotechnology Center, Universita di Torino, 10126 Torino, Italy

ABSTRACT: Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is an intracellular biopolyester synthesized by various bacteria. Polyhydroxyalkanoate granule-binding protein (PhaP), a natural biomacromolecule symbiotic with PHBHHx, is talented to be steadily adsorbed to PHBHHx matrix through via hydrophobic interactions. In this study, PHBHHx nanoparticles (NPs) and iRGD peptide fused PhaP (iRGD-PhaP) were used in conjunction to build a specific drug delivery system for targeted accumulation and tissue penetration in prostate tumor. A proper presentation and high surface density of iRGD could be ensured within 1 h through a convenient co-incubation method using a PhaP-mediated modification strategy. iRGD-PhaP-NPs showed a satisfactory particle size (182.9 ± 4.9 nm) and slightly negative surface charge (-17.2 ± 0.3 mV), with a uniformly spherical shape. In human prostate cancer cell line PC3, iRGD-PhaP-

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NPs displayed remarkably improved cellular uptake compared to naked NPs, which was attributed to iRGD receptor-mediated active endocytosis. Enhanced targeted accumulation and retention of iRGD-PhaP-NPs to prostate tumor were found in both the ex vivo tumor spheroid assay and in vivo real-time imaging. Moreover, slices of the tumor deep region demonstrated the favorable tumor penetration ability of iRGD-PhaP-NPs after intravenous administration. These results highlight the specificity and efficiency of iRGD-PhaP-NPs in future clinical use.

KEYWORDS: PHBHHx nanoparticles; PhaP; iRGD; Tumor targeting and penetration 1. INTRODUCTION Over the past decades, researchers have designed a variety of nano-scaled targeted drug delivery systems (TDDSs) with hopes of application in therapies against solid carcinoma.1-2 Wherein the biodegradable polymers based nanoparticles (NPs) have gained widespread attention due to the satisfactory biocompatibility, formability and capacity for controlled drug release.3-4 Certain receptors and antigens are known to be aberrantly upregulated in tumors and nearby tissues, such as gastrin releasing peptide receptors (GRPR), vascular endothelial growth factors (VEGF), protease-specific membrane antigen (PSA), matrix metalloproteinases (MMPs) and so on.5-7 Accordingly, TDDSs are commonly constituted by presenting ligands or molecules with affinity to those receptors or antigens on the surface of polymeric NPs, which enable the selective accumulation of chemotherapeutic drugs in solid tumors. Nevertheless, the biomedical application of traditional TDDSs is still restricted by several unsatisfactory properties: i) The surface functionalization of polymeric NPs is mainly realized via covalent crosslinking reaction, which can be extremely cumbersome and time-consuming.8-11 Moreover, residues of the crosslinking reagents may lead to unpredictable adverse effects.12-13 ii) The frequently used


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native ligands, such as epidermal growth factor (EGF) which could specifically recognize the epidermal growth factor receptor (EGFR), can potentially trigger unwelcome downstream physiological responses.14 iii) The dense tumor parenchyma and high interstitial fluid pressure form a strong barrier to inhibit the transcapillary transport of TDDS and block the penetration of NPs into the tumor parenchyma.15-16 Polyhydroxyalkanoates (PHAs) are a class of microbially synthesized biopolyesters, which serve as intracellular carbon source.17-18 Due to a series of excellent features, including thermoprocessability, biocompatibility, biodegradability and non-toxicity of the degradation products, PHAs have been extensively studied for in vivo application as implantable devices.19-20 It is noteworthy that PHAs have also been increasingly exploited as polymeric nanocarriers for hydrophobic drug delivery, which benefit from their aliphatic side-chains.21-22 Our group had successfully prepared NPs based on PHBHHx, a new generation of commercial PHA materials, for encapsulating the PI3K inhibitor TGX221 and Rapamycin. Drug-loaded PHBHHx NPs exhibited improved sustained release performance and stronger anti-proliferation effects in human prostate cancer cell line PC3 than either drug-loaded PLA NPs or free inhibitors.23-24 Natural PHA granules are surrounded by multiple proteins, including the PHA depolymerase (PhaZ), PHA regulatory protein (PhaR) and PHA granule binding protein (PhaP).25 PhaP is an amphiphilic protein with a hydrophobic moiety that binds with PHA granules and a hydrophilic moiety that is exposed to the intracellular aqueous environment.26-27 Taking advantage of PhaP’s natural features, a low-cost protein purification system was built by fusing the target protein to PhaP via a self-cleaving linker, and PHA NPs were then used to adsorb fusion proteins from cell lysates.28 Yao et al. fused the human α1-acid glycoprotein (hAGP) and human epidermal growth factor (hEGF) with PhaP. The fusion proteins were anchored on PHA NPs to achieve targeted


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delivery.29 An immune costimulatory molecule, B7-2, was fused with PhaP and presented on PHA NPs, which successfully induced the activation and expansion of T cells in vitro.30 Overall, a novel and convenient approach for surface functionalization of PHA NPs, distinguished from the generally applied chemical crosslinking method, has been established and continuously ameliorated. Particularly, PhaP was also found to adsorb onto the surface of other common hydrophobic polymers such as poly (lactic-co-glycolic acid) (PLGA), which provides a broader application space for PhaP-mediated surface functionalization.31 The artificial tumor-homing peptide iRGD has been identified to exhibit a tumor-specific vascular extravasation and tissue penetration activity.32 iRGD preferentially recognizes integrin αvβ3/5 via the RGD motif, and then subjected to a tumor-derived proteolytic cleavage to reveal the active C-end Rule motif, which binds to neuropilin-1 (NRP-1) and leads to extravasation and penetration.9-11 The integrin αvβ3/5 is specifically expressed on the vascular endothelial cells in angiogenesis.33 The NRP-1 is overexpressed in various solid tumor cells such as the colorectal adenocarcinomas,34 pancreatic adenocarcinomas,35 and prostate tumor cells.36 Hence, a variety of TDDSs using iRGD as ligand have been developed to encourage the targeted accumulation and tissue penetration of chemotherapeutic drugs in solid tumors.37-38 In the present work, we fused iRGD to PhaP via a designed linker, and the fusion protein iRGD-PhaP was expressed and purified from recombinant E. coli. PHBHHx NPs were then surface-functionalized with iRGD-PhaP using the abovementioned approach to construct a specific TDDS (Scheme 1). The interaction between iRGD-PhaP and PHBHHx NPs, as well as the physicochemical properties of iRGD-PhaP-NPs were fully characterized. Additionally, the in vitro cellular uptake of iRGD-PhaP-NPs in human prostate cancer cell line PC3 was investigated.


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We further evaluated the targeted accumulation and tissue penetration both ex vivo and in vivo using a PC3 xenograft mouse model.

Scheme 1. Illustration of the development and functional verification of the specific TDDS based on microbially synthesized PHBHHx biopolyester and iRGD peptide fused PhaP (iRGDPhaP-NPs). 2. MATERIALS AND METHDOS 2.1 Plasmid, primers and reagents


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Table 1 displayed details of plasmid and primers used in this study. PHBHHx was produced and isolated as described previously.39 Poly (vinyl alcohol) (PVA) was supplied by Sigma-Aldrich (MO, USA). Rhodamine B (RB), 4,6-diamidino-2-phenylindole (DAPI), and 1,1’-dioctadecyl3,3,3’,3’-tetramethyl indotricarbocyanine iodide (DiR) were obtained from Biotium (CA, USA). Table 1. Plasmid and primers used in this study. Name

Genotype, description or sequence

Source

Ampr, pTwin2 derivative containing the PhaP operon from Ralstonia eutropha H16

Tsinghua University

Nde Ⅰ-His-PhaP

5'-AACATATGCACCACCACCACCACCACAATATGGACGT GATCAAGAGCTTTACC-3'

This study

PhaP-iRGD-BamH Ⅰ

5'-ATGGATCCTCAGCATTCCGGACCTTTGTCACCACGGCACAGTTCGAGACCGTTGTTACC-3'

This study

Plasmids pTwin1-EPhaP Primers

2.2 Cell lines and animals Human prostate cancer cell line PC3 was placed in a humidified atmosphere comprising 5% CO2 at 37 °C, and cultured with RPMI 1640 medium (Invitrogen, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% antibiotics. SPF BALB/c nude mice (Male, 3 weeks old) were purchased from Vital River Laboratory Technology (Beijing, China) and feed at the Department of Experimental Animals, Xi’an Jiaotong University (Shaanxi, China). Mice were treated and sacrificed under the permission of the ethics committee of Xi’an Jiaotong University (Shaanxi, China). 2.3 Construction, preparation and structural analysis of iRGD-PhaP The gene-specific forward primer (Nde Ⅰ-His-PhaP) and corresponding reverse primer (PhaPiRGD-BamH Ⅰ) were designed to clone PhaP gene from the plasmid pTwin1-EPhaP to generate the target sequence (His tag–PhaP-iRGD). The final recombinant plasmid was constructed by inserting the digested PCR products into the pTwin1 vector. The expression and purification of


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iRGD-PhaP followed the routine procedure.40 The purity of the isolated iRGD-PhaP was further confirmed by LC-MS (ACQUITY UPLC H-Class/SQD2, Waters, USA), and the material was condensed for further usage. To visualize the recombinant protein for further analysis, a threedimensional model of iRGD-PhaP was constructed by using the solution structure of PhaP (PDB code: 5IP0) as template.27, 41 The initial 3D structure was built and optimized by the molecular dynamics simulation with Modeller 9.19.42 The energy minimization was conducted to improve the physical realism and side-chain accuracy on the YASARA Energy Minimization Server (http://www.yasara.org/index.html).43 All visual inspections and generation of figures were carried out in PyMol 2.1. 2.4 Fabrication and characterization of PHBHHx NPs A modified emulsification/solvent evaporation method was used to prepare PHBHHx NPs as described previously.40 The particle size, polydispersity index (PDI) and surface zeta potential were measured by a Zetasizer Nano ZS (Malvern Instruments, UK). The morphology of NPs was analyzed by a transmission electron microscopy (TEM; H-600, Hitachi, Japan), and individual NP was observed by using a field emission cryo-transmission electron microscopy (FECTEM; Talos F200C, FEI NanoPorts, USA). The surface hydrophobicity of NPs was characterized by a contact angle measurement method.40, 44 All the observations were also performed after the NPs were surface-functionalized with iRGD-PhaP. 2.5 Surface functionalization of PHBHHx NPs with iRGD-PhaP 0.5 mg of PHBHHx NPs and 125 µg of iRGD-PhaP were incubated in l mL of PBS at room temperature, pH 7.4 for 1, 2, 6, 12 h, respectively. After this serial of different incubation times, the mixture was centrifugated to separate the free iRGD-PhaP in the supernatant from the precipitate. The supernatant was analyzed using a bicinchoninic acid (BCA) method to calculate


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the binding rate and saturated binding amount. The precipitate was washed and resuspended in PBS to obtain iRGD-PhaP-NPs. Both the supernatant and precipitate were investigated by 10% acrylamide SDS-PAGE and western blotting. Furthermore, PHBHHx NPs were also incubated with iRGD-PhaP at room temperature, pH 6.0 to assess the tolerance of this binding process to a tumor-liked acidic environment. The binding rate was characterized by the variation of free iRGD-PhaP with the incubation time. The saturated binding amount was calculated according to equation (1). 𝐄𝐪. (𝟏) 𝐒𝐚𝐭𝐮𝐫𝐚𝐭𝐞𝐝 𝐛𝐢𝐧𝐝𝐢𝐧𝐠 𝐚𝐦𝐨𝐮𝐧𝐭 (µ𝐠/𝐦𝐠) =

(𝟏𝟐𝟓 − 𝐚𝐦𝐨𝐮𝐧𝐭 𝐨𝐟 𝐟𝐫𝐞𝐞 𝐢𝐑𝐆𝐃 − 𝐏𝐡𝐚𝐏) µ𝐠 𝟎. 𝟓 𝐦𝐠

And the binding efficiency (BE%) was calculated using equation (2). 𝐄𝐪. (𝟐) 𝐁𝐄 (%) =

(𝟏𝟐𝟓 − 𝐚𝐦𝐨𝐮𝐧𝐭 𝐨𝐟 𝐟𝐫𝐞𝐞 𝐢𝐑𝐆𝐃 − 𝐏𝐡𝐚𝐏) µ𝐠 × 𝟏𝟎𝟎% 𝟏𝟐𝟓 µ𝐠

The surface density of iRGD-PhaP was calculated by dividing the number of iRGD-PhaP molecules by the average number (N) of NPs. Equation (3) was applied to calculate the value of N.8, 45 𝐄𝐪. (𝟑) 𝐍 = (𝟔 × 𝐦)⁄(𝛑 × 𝐃𝟑 × 𝛒)

Where m is the weight of NPs; D is the mean diameter of NPs; ρ is the density of NPs. 2.6 In vitro tumor cellular uptake Human prostate cancer cell PC3 were seeded at 4 × 104 cells/well into 24-well laser confocal special culture plates, and cultured overnight. Afterwards, the cells were treated by RB-loaded NPs and iRGD-PhaP-NPs at 100 µg/mL for 1 h, respectively. Moreover, a group of PC3 cells were firstly treated by 200 µg/mL of free iRGD-PhaP for 1 h, then by 100 µg/mL of RB-loaded iRGD-PhaP-NPs for another 1 h. Rinsed with PBS thrice to remove extracellular NPs and fixed with 4% paraformaldehyde for 15 min, cells were ready to be stained with DAPI. Eventually, a


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laser scanning confocal microscope (LSM700, ZEISS, Germany) analysis was applied on stained cells to evaluate the in vitro cellular uptake. The cellular fluorescence intensities were quantified using Image-Pro Plus 6.0. 2.7 Ex vivo tumor spheroid assay 1 × 106 PC3 cells were inoculated subcutaneously in the lateral limb of nude mouse (4 weeks old) to establish a prostate tumor xenograft mouse model. Two to three weeks after implantation, tumor masses of different sizes, ranging from 100 mm3 to 200 mm3, were carefully resected. The obtained PC3 tumor spheroids were temporarily cultured in 1640 medium and incubated with RB-loaded NPs and iRGD-PhaP-NPs at 100 µg/mL for 1 h, respectively. Afterwards, the tumor spheroids were rinsed with PBS thrice, then subjected to a fluorescence imaging to evaluate the targeted accumulation. The tumor spheroids were continuously cultured without any NPs, and fluorescence images were acquired at 1, 2, 3, 4 h post incubation to assess the tumor retention. 2.8 In vivo real-time imaging PC3 xenograft mouse model was prepared as mentioned earlier. Two weeks after inoculation, mice were treated by the equal amount of DiR-loaded NPs and iRGD-PhaP-NPs via tail vein injection, respectively. The fluorescence distributions were captured at 1, 2, 6, 24 h post injection through a in vivo imaging system (IVISSPE, PE, USA) to trace the in vivo targeted accumulation in the tumor. Subsequently, major organs from the treated mice including the hearts, livers, spleens, lungs, and kidneys, as well as the tumors were collected for fluorescence imaging. Other mice were intratumorally injected by the equal amount of RB-loaded NPS and iRGD-PhaP-NPs at a fixed depth of needle insertion, respectively. The fluorescence images were acquired at 0, 1, 3, 5 h following injection to investigate the tumor retention. 2.9 Evaluation of tumor penetration


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As the tumor volume reached 300 mm3, the mice were treated by the equal amount of RB-loaded NPs and iRGD-PhaP-NPs via tail vein injection, respectively. At 24 h post injection, the tumors were excised and washed, then cut into slices at different layers from the edge region to the deep region of the tumors. The slices were stained with DAPI and observed under a laser confocal microscopy (LSM700, ZEISS, Germany). 2.10 Statistical analysis All the data were presented as means ± SD. Statistical analyses were performed by one-way ANOVA followed by Bonferroni tests. P < 0.05 was considered to be a significant difference. 3. RESULTS AND DISCUSSION 3.1 Construction, preparation and structural analysis of iRGD-PhaP iRGD was fused to the C-terminus of PhaP via a rigid protein linker (NNGNNGLEL) so as to frame a dual-functional fusion protein iRGD-PhaP, and a 6 x His tag was hired to enable the further purification (Figure 1a). The construction of the recombinant plasmid for iRGD-PhaP expression followed routine procedures, and the plasmid map is displayed in Figure 1b. The final recombinant plasmid was confirmed by restriction-enzyme digestion (Figure 1c, Lanes 1&2) and PCR (Figure 1c, Lane 3) followed by DNA sequencing (Data not shown). The verified plasmid was transformed into E. coli BL21 (DE3) and the expression of iRGD-PhaP was induced with IPTG. Compared with the wild-type (Figure 1d, Lane 1), a novel major band at approximately 15 KD was detected in the cleared lysate of recombinant E. coli BL21 (DE3) (Figure 1d, Lane 2). Moreover, there was just one band at 15 KD in the purified fraction (Figure 1d, Lane 4), indicating an excellent purification effect. The molecular weight of this band was 15340 ± 1.3 according to LC-MS (Figure 1e), which matched the estimated value of iRGD-PhaP.


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Figure 1. Construction, preparation and structural analysis of the iRGD-PhaP fusion protein. (a) The composition of iRGD-PhaP. (b) Detailed map of the plasmid used for the expression of iRGD-PhaP. (c) Agarose gel electrophoresis of the recombinant plasmid. M, DNA marker; Lane 1, double digested with Nde Ⅰ and EcoR Ⅴ; Lane 2, double digested with BamH Ⅰ and EcoR Ⅴ; Lane 3, recombinant plasmid was subjected a PCR with primers in Table 1, showing the 425 bp insert encoding iRGD-PhaP CDS. (d) Acrylamide SDS-PAGE of iRGD-PhaP. M, marker; Lane 1, lysate of induced wild-type bacterium; Lane 2, lysate of induced recombinant bacterium; Lane 3, washing fraction of the lysate from the induced recombinant bacterium in the purification process; Lane 4, purified iRGD-PhaP eluted from the nickel-affinity column. (e) LC-MS confirmation of iRGD-PhaP. (f) Three-dimensional structure of iRGD-PhaP (red, iRGD; purple, rigid protein linker; green, PhaP; yellow, His-Tag). (g) Superposition of iRGD-PhaP and independent PhaP (blue: independent PhaP).


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It has been reported over thirty years ago that the two hydrophobic domains of PhaP are indispensable for the anchoring of PhaP to natural PHA granules.26 Recent crystallographic structural insights revealed the hydrophobic side of PhaP, which was critical to the this natural adsorption.

27

Since the functions of PhaP and iRGD both depend on their structures, the 3D

structure of iRGD-PhaP was constructed by homology modeling to determine whether the two components affect each other. Molecular dynamics simulation was performed to improve the primary structure. As shown in Figure 1f, iRGD (red) did not spatially entangled with PhaP (green), which confirmed the structural integrity of iRGD in fusion protein. When superimposed, the PhaP fragment (green) in fusion protein was basically consistent with the independent PhaP (blue), which confirmed the structural integrity of PhaP. More importantly, the direction which iRGD protruded in was opposite to the hydrophobic side of PhaP due to the rigid protein linker (Figure 1f&g). This conformation not only ensured the adsorption of PhaP to PHBHHx NPs, but also facilitated the proper presentation of iRGD to exert its function. 3.2 Synthesis and physicochemical properties of PHBHHx NPs A well-established emulsification solvent evaporation method was applied in this study to synthesize PHBHHx NPs.21, 23-24, 30 Due to the Marangoni interface effect, the drop-by-drop mix of the oil phase (PHBHHx/chloroform) and aqueous phase (PVA/water) formed a uniform oil/water emulsion under ultrasound agitation. With the evaporation of the organic solvent, PHBHHx droplets gradually solidified into NPs via the hydrophobic interaction.46 PVA was used as an emulsifier in this synthesis process to stabilize the emulsion and prevent aggregation, and it should eventually be removed as much as possible to avoid impairing the surface properties of NPs.40, 47-48


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DLS analysis revealed that the mean hydrodynamic diameter of PHBHHx NPs was 167.1 ± 3.6 nm, with a negative charge at -24.8 ±1.7 mV (Table 2). Polymeric NPs with a slight negative charge and hydrodynamic diameter less than 200 nm were more inclined to target tumor tissue, which demonstrated the potential value of PHBHHx NPs.49-50 Moreover, a relatively low PDI value (0.02 ± 0.03) of the PHBHHx NPs was measured, which was consistent with their narrow distribution (Figure 2a). Morphological observations by TEM and FECTEM both showed that the PHBHHx NPs were typically spherical and well dispersed without any aggregation (Figure 2c&e). The average diameter of the PHBHHx NPs according to TEM photograph was about 120 nm, which was remarkably smaller than DLS result. This inconsistency can be explained by the fact that the PHBHHx NPs were completely dehydrated during TEM imaging, but fully hydrated during DLS analysis.10 The deionized water drop on the thin film of PHBHHx NPs angled at 27.18 ± 1.74°, indicating a partially wetted surface (Figure 2g&i).51 Even though this surface of PHBHHx NPs can barely be defined as hydrophobic, the hydrophobic side of PhaP would still attach onto it to decrease the entropy of the complex system due to the superior emulsification property of PhaP.27-30 The results of our subsequent experiments also verified this point of view. Table 2. Characterization of PHBHHx nanoparticles. Nanoparticles

Size (nm)

Polydispersity index

Zeta potential (mv)

Saturated binding amount (µg/mg)

Binding efficiency (%)

Surface density

NPs

167.1 ±3.6

0.02 ±0.03

-24.8 ±1.7

-

-

-

iRGD-PhaP-NPs

182.9 ±4.9

0.16 ±0.01

-17.2 ±0.3

174 ±12

70 ±5

1383 ±95


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Figure 2. Size distribution of NPs (a) and iRGD-PhaP-NPs (b). TEM photographs of NPs (c) and iRGD-PhaP-NPs (d). FECTEM photographs of NPs (e) and iRGD-PhaP-NPs (f). Still photographs of the deionized water drop on NPs thin films (g) and iRGD-PhaP-NPs thin films (h). Contact angles of NPs and iRGD-PhaP-NPs (i), **p < 0.01. 3.3 Surface functionalization of PHBHHx NPs with iRGD-PhaP A convenient co-incubation method was performed to prepare the specific TDDS (iRGD-PhaPNPs) via the natural adsorption between PhaP and PHA material.26-30 After 1 hrs incubation, a considerable amount of iRGD-PhaP was detected in the precipitate of PHBHHx NPs (Figure 3a, Lane 5), demonstrating that iRGD-PhaP could be tightly bound to PHBHHx NPs even suffered multiple washing. With the extension of the incubation time, there was no significant alteration in the amount of free iRGD-PhaP in the supernatant (Figure 3a, Lanes 1-4), which revealed that


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this binding process would reach saturation within 1 h. We herein designated the incubation time as 1 h and the resulting iRGD-PhaP-NPs were characterized as mentioned above. The saturated binding amount and the surface density of iRGD-PhaP on the PHBHHx NPs are listed in Table 2. As expected, the surface functionalization with iRGD-PhaP slightly enlarged the diameter and surface potential of NPs to 182.9 ± 4.9 nm and -17.2 ± 0.3 mV, respectively. The FECTEM photograph of iRGD-PhaP-NPs confirmed a palpable protein corona of iRGD-PhaP was ‘honoured’ on the surface of the PHBHHx matrix due to the difference in electron density (Figure 2f). The correct presentation of the fusion protein was achieved, and the hydrophilic side of iRGD-PhaP was exposed outwards to significantly improve the surface hydrophilic of the iRGD-PhaP-NPs (Figure 2h&i).

Figure 3. (a) Acrylamide SDS-PAGE and WB of the supernatant and precipitate when the NPs were incubated with iRGD-PhaP at room temperature, pH 7.4 for different lengths of time. M, marker; Lanes 1-4, supernatant after 1, 2, 6, 12 hrs incubation, respectively; Lanes 5-8, precipitate after 1, 2, 6, 12 hrs incubation, respectively. (b) Acrylamide SDS-PAGE and WB of the supernatant (Lane 1) and precipitate (Lane 2) when the NPs were incubated with iRGD-PhaP at room temperature, pH 6.0 for 1 h. Generally, the surface functionalization of polymeric NPs was mainly achieved through covalent crosslinking between reactive groups on the surface of polymeric NPs and functional


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peptides.9-11 The crosslinking reaction typically takes at least 8 h to ensure conjugation efficiency, resulting in a considerable loss of loaded drugs.8 Besides, the introduction of a series of crosslinking reagents, such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), may cause adverse reactions in future biomedical applications.12-13 In this regards, the PhaP-mediated surface functionalization was much more convenient and secure. Since no reactive groups are required, the functionalization efficiency was perceptibly higher. More importantly, Figure 4b demonstrated that this modification strategy resulted in NPs that can withstand the acidic environment typical of tumors, and was thus suitable for targeted accumulation and tissue penetration. 3.4 In vitro tumor cellular uptake Improved cellular uptake of TDDS by the targeted cells is a vital indicator for the increase of therapeutic efficacy. The ability of cellular internalization of NPs and iRGD-PhaP-NPs was investigated under a laser scanning confocal microscope, using the integrin αvβ3/5 and NRP-1 highly overexpressed PC3 cells as a cell model and RB as a fluorescent probe.5, 33, 52 A modest intensity of intracellular fluorescence was noticed in PC3 cells incubated with RB-loaded NPs, which indicated the effective cellular uptake as a result of the appropriate diameter and surface potential of NPs (Figure 4).49-50 As predictable, the intracellular fluorescence intensity of the PC3 cells treated with iRGD-PhaP-NPs was about 1.78-fold higher than with undecorated NPs, which confirmed that the surface functionalization with iRGD-PhaP did enhance the internalization of NPs by the PC3 prostate cancer cells. Additionally, a competition assay was performed to explore the mechanism of the intracellular uptake of iRGD-PhaP-NPs. The heightened cellular internalization of iRGD-PhaP-NPs was drastically blocked by the pre-treatment with free iRGDPhaP, suggesting that the iRGD-PhaP-NPs were ingested by PC3 cells through iRGD receptor-


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mediated active endocytosis. One rational suppose for this phenomenon is that PC3 cells might be able to secrete a protease able to expose the C-end Rule motif of iRGD-PhaP, which in turn would lead to binding between the CendR motif and NRP-1, and finally trigger cellular internalization. Although direct evidence is still scarce, similar proteolytic cleavage processes were repeatedly reported at the cellular level.53-54 In addition, the fluorescence images also revealed that iRGD-NPs could be transported into the nucleus, which makes it a potential nanocarrier for intra-nuclear drug delivery.

Figure 4. In vitro uptake of NPs, iRGD-PhaP-NPs and iRGD-PhaP-NPs in the presence of free iRGD-PhaP by PC3 tumor cells. (a) Fluorescence images. Scale bars, 10 μm. (b) Semiquantitative fluorescence intensity per cell. Comparison of the intracellular fluorescence intensity with the NPs treated group and the iRGD-PhaP pre-blocked group, **p < 0.01.


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3.5 Ex vivo tumor spheroid assay Intact solid tumors with different dimensions were harvested from the PC3 xenograft mice as ex vivo tumor spheroids models, and immediately incubated with RB-loaded NPs and iRGD-PhaPNPs for 1 h. Since the tumor tissues cultured in vitro were able to maintain physiological activity for a short time,55-56 iRGD mediated tumor targeted accumulation and retention ought to be triggered in tumor spheroids treated with iRGD-PhaP-NPs. The fluorescence data graphically displayed that the iRGD-PhaP-NPs treated group was 1.62-fold brighter than NPs treated group in various sizes of tumor spheroids (Figure 5a&b). The tumor spheroids were further cultured without any NPs to evaluate the tumor retention. With prolonged culture time, the intratumoral fluorescence intensities were reduced in both the NPs and iRGD-PhaP-NPs treated groups (Figure 5a&c), due to the redistribution of NPs to the medium. Nevertheless, the reduction of intratumoral fluorescence intensity in the iRGD-PhaP-NPs treated group was obviously slower than in the NPs treated group. Furthermore, about 51% of the intratumoral fluorescence signal was still presented in the iRGD-PhaP-NPs treated tumor spheroids at 4 h post incubation, while only around 35% remained in the NPs treated tumor spheroids. These results confirmed the improved targeted accumulation and retention of iRGD-PhaP-NPs in tumor spheroids ex vivo, which benefited from the upgraded internalization by tumor cells. Notably, this attractive property did not show a tumor size-dependent pattern, implying the possible applicability of iRGD-PhaP-NPs to early prostate cancer.


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Figure 5. (a) Fluorescence images of harvested tumor spheroids with different sizes treated with NPs and iRGD-PhaP-NPs for 1 h, respectively. (b) Semi-quantitative average fluorescence signals of tumor spheroids after 1 hrs incubation, **p < 0.01. (c) Relative fluorescence intensity of the tumor spheroids at 1, 2, 3, 4 h post incubation, respectively. Comparison of the intratumoral fluorescence intensity with the NPs treated group, *p < 0.05, **p < 0.01. 3.6 In vivo real-time imaging The immunogenicity of iRGD-PhaP-NPs was first investigated before in vivo application. The immunohistochemistry (IHC) of mouse immunoglobulin G (IgG) in major organs suggested no significant immune response even if the dose of iRGD-PhaP-NPs was as high as 500 mg/kg (Data not shown). The in vivo tumor targeted accumulation of iRGD-PhaP-NPs in prostate tumor xenograft mice was evaluated through applying a near-infrared (NIR) fluorescent dye DIR as indicator.57 The fluorescence signal could be detected in the tumor site after injecting for 6 h and


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became stronger after 24 h in both the NPs and iRGD-PhaP-NPs administered tumor-bearing mice (Figure 6a). An apparently higher level of tumor-fluorescence was observed in the iRGDPhaP-NPs treated tumor-bearing mice at all time points. However, the majority of NPs were removed from circulation by the liver and only a modest level of tumor-fluorescence was exhibited in the NPs treated nude mice due to the relatively more hydrophobic surface and absence of specificity. The major organs and tumors were harvested at 24 h post intravenous administration. In the iRGD-PhaP-NPs administered tumor-bearing mice, a significantly weaker fluorescence signal (about 30% weaker than the NPs treated mice) was detected in the liver and a obviously stronger fluorescence signal (almost 2.05-fold stronger than the NPs treated mice) was detected in the tumor of (Figure 6b&c). These results demonstrated the surface functionalization with iRGD-PhaP did ameliorate the targeted accumulation of NPs in prostate tumor.

Figure 6. (a) In vivo real-time imaging of the PC3 xenograft mice intravenously treated with NPs and iRGD-PhaP-NPs at the time point of 1, 2, 6, 24 h, respectively. The dotted circles indicate the tumor site. (b) Fluorescence imaging of major organs and tumors at 24 h post-injection. (c)


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Semi-quantitative average fluorescence signal in each organ and the tumor. Comparison of the average fluorescence intensity with the NPs treated group, **p < 0.01. Furthermore, intratumoral administration was used to characterize the in vivo tumor retention of iRGD-PhaP-NPs. Within 1 h after intratumoral administration of RB-loaded NPs, the fluorescence signal within the tumor site was sharply attenuated, and eventually could practically no longer be observed after 5 h post-injection (Figure 7a). One plausible explanation for this observation is that NPs were transported into blood circulation through the abundant microvessels inside the tumor xenograft, and could consequently no longer be traced due to the shading by subcutaneous tissues. By contrast, the decay rate of the intratumoral fluorescence signal after intratumoral administration of RB-loaded iRGD-PhaP-NPs was significantly slower, and there was still around 42% of residual fluorescence at 5 h. Thus, a considerable amount of iRGD-PhaP-NPs remained in the tumor parenchyma instead of being eliminated by the transcapillary transport, since surface functionalization with iRGD-PhaP increased the tumor cellular uptake of NPs.


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Figure 7. (a) In vivo real-time imaging of the PC3 xenograft mice intratumorally injected with NPs and iRGD-PhaP-NPs at the time point of 0, 1, 3, 5 h, respectively. (b) Relative intratumoral fluorescence intensity at 1, 3, 5 h after administration, respectively. Comparison of the relative intratumoral fluorescence intensity with the NPs treated group, **p < 0.01. 3.7 Evaluation of tumor penetration Slices of the tumor edge and tumor deep regions (3 mm from the tumor edge) were prepared separately to assess the tumor penetration of NPs and iRGD-PhaP-NPs. At 24 h after intravenous administration, the fluorescence signal indicated the accumulation of iRGD-PhaP-NPs in the tumor edge region was obviously more intense than the NPs (Figure 8a). As shown in Figure 8b, only the traces of NPs could reach the tumor deep region, since the penetration of NPs was extremely restricted by the dense tumor parenchyma and high interstitial fluid pressure.58 By


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contrast, the fluorescence of iRGD-PhaP-NPs in the tumor deep region was just slightly less than in the tumor edge region, suggesting that the surface functionalization with iRGD-PhaP did overcome these barriers and facilitate the tumor penetration of NPs.

Figure 8. Fluorescence signal of NPs and iRGD-PhaP-NPs accumulated in the slices of tumor edge region (a) and tumor deep region (b). Scale bars, 50 μm. 4. CONCLUSION A specific TDDS based on microbially synthesized PHBHHx biopolyester and iRGD peptide fused PhaP was successfully developed for facilitating efficient drug delivery to solid tumors. This novel and convenient system consists of PHBHHx NPs, which were proven to be a highly versatile nanocarrier for various hydrophobic drugs, and PhaP, which was a bio-linker to stably decorate the NPs with the tumor-homing peptide iRGD. The iRGD-PhaP fusion protein could correctly and efficiently presented on PHBHHx NPs within 1 h without any tedious operations and risks of chemical-related side effects. The resulted iRGD-PhaP-NPs displayed a regularly spherical shape with a desirable particle size (182.9 ± 4.9 nm) and negative surface charge (-17.2 ± 0.3 mV), which was beneficial for cellular uptake. Compared with naked NPs, iRGD-PhaPNPs exhibited an improved internalization in the human prostate cancer cell line PC3. Both ex vivo and in vivo experiments demonstrated that iRGD-PhaP-NPs had a significantly enhanced


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targeted accumulation and tissue penetration ability in the prostate tumor models. Although the anti-tumor efficacies of iRGD-PhaP-NPs in prostate tumor or other types of solid tumors after drug loading still need further evaluation, these results definitely indicate the promising potential of iRGD-PhaP-NPs as a nanocarrier of anti-tumor drugs for prostate cancer therapy. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Fan Fan: 0000-0002-6587-0416 Xiaoyun Lu: 0000-0003-3284-4729 Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Nos. 81172170 and 81371288) and the Fundamental Research Funds for the Central Universities from Xi’an Jiaotong University. We are also grateful for assistance from Prof. Guoqiang Chen (Lab of Microbiology, Department of Biological Science and Biotechnology, Tsinghua University). REFERENCES (1) Grever, M. R.; Schepartz, S. A.; Chabner, B. A. The National Cancer Institute: Cancer Drug Discovery and Development Program. Semin. Oncol. 1992, 19 (6), 622-638. (2) Karthikeyan, R.; Koushik, O. S. Nano Drug Delivery Systems to Overcome Cancer Drug Resistance - A Review. J. Nanomed. Nanotechnol. 2016, 7 (3), 1-9.


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