Smart pH-sensitive nanoassemblies with cleavable PEGylation for tumor targeted drug delivery

A new acidly sensitive PEGylated polyethylenimine linked by Schiff base (PEG-s-PEI) was designed to render pH-sensitive PEGylation nanoassemblies through multiple interactions with indomethacin and docetaxel (DTX). DTX nanoassemblies driven by PEG-s-PEI thus formulated exhibited an excellent pH-sensitivity PEGylation cleavage performance at extracellular pH of tumor microenvironment, compared to normal tissues, thereby long circulated in blood but were highly phagocytosed by tumor cells. Consequently, this smart pH-sensitive PEGylation cleavage provided an efficient strategy to target tumor microenvironment, in turn afforded superior therapeutic outcome in anti-tumor activity.

we developed a new acidly sensitive PEGylation cleavable PEI linked by Schiff base which is used to render pH-sensitive PEGylated NAs through multiple interactions with small molecule drugs mediated self-assembly in this study. Nanoparticles thus produced, with facile material synthesis, high drug loading capacity, desirable therapeutic benefits, low toxicity for intravenous application and pH-triggered deshielding of PEG, can serve as efficient and tumor environment targeting nanocontainers for anti-cancer drugs, and conducive to clinical transformation of PEGylation cleavable nanotherapeutics.
Hydrolysis of PEG-s-PEIs were carried out that half-life times of PEG-s-PEIs were more than 5 h at pH 7.4, but less than 25 min at lower pH. The half-life times of PEG-s-PEI-1 and PEG-s-PEI-2 were 90 min and 2 h at pH 6.5, respectively ( Fig. S2a,b). And the cytotoxicity of PEG-s-PEI-2 against HepG2 was significantly decreased than PEI, either in pH 6.5 or pH 7.4 mediums (Fig. S2c,d).

Computational design and assembly of DTX/IND/PEG-s-PEI. Initially, indomethacin (IND), a widely
used nonsteroidal anti-inflammatory drug (NSAID), was utilized as guest molecular to dock into PEG-s-PEI or PEI. IND and PEG-s-PEI displayed a little lower intermolecular energy compared to IND and PEI interaction. However, it is significantly higher than that of other combinations in IND/PEG-s-PEI system (Fig. 2a). The lowest energy conformations of IND within PEG-s-PEI chains or PEI chains after the docking calculations in 3D conformation were both exactly "embraced" within big pockets with H-bonding and hydrophobic areas formed by the sidechains and backbone atoms of PEG-s-PEI or PEI (Fig. 2b,c). Based on computational results, self-assemblies of DTX, IND and PEG-s-PEI with various DTX/IND/ PEG-s-PEIs weight ratios were conducted by dialysis against deionized water using methanol as the common solvent 40 . Independent of DTX/IND/Polymer weight ratio, spherical nanoassemblies were assembled with monodispersed distribution profiles (Fig. 3a). The hydrodynamic diameter (D h ) of assemblies increased when the PEG/PEI molar ratio increased (Fig. 3b), while ζ-potential decreased (Fig. 3c). HPLC quantification revealed a dramatic enhancement in DTX loading content when drug feeding ratios of DTX/IND/PEG-s-PEIs increased (Fig. 3d), while IND loading contents were correspondingly reduced (Fig. S3). Compared with DTX/IND/PEI NAs, the hemolysis ratio of DTX/IND-PEG-s-PEI-1, DTX/IND/PEG-s-PEI-2 and DTX/IND/PEG-b-PEI was lowered to 11.34 ± 1.56, 3.231 ± 0.822 and 1.561 ± 0.351, respectively (Table S2). pH triggered PEGylation cleavage with potential shift. DTX/IND/PEG-s-PEI NAs at weight ratio of 10:10:10 were employed to study pH triggered PEGylation cleavage (Fig. 3e). At pH 5.5 and 6.5, DTX/IND/ PEG-s-PEI NAs formed significantly surface potential shifting in 120 min, while stably retained low surface charge for the whole 2 h time period at pH 7.4. On the contrary, stable surface charge of DTX/IND/PEI NAs was maintained at 60 mV in all buffers even after 24 h later. In Vitro Release and In Vivo Pharmacokinetic Studies. Under in vitro conditions, DTX/IND/ PEG-s-PEI NAs showed considerably rapid release at pH 5.0, slower release at pH 6.5, and further slower at pH 7.4 (Fig. 5a). Comparatively, no difference was found when DTX/IND/PEG-b-PEI NAs were employed to be released in vitro conditions (Fig. 5a). Subsequently, in vivo pharmacokinetic study carried out (Fig. 5b), the t 1/2 of DTX/IND/PEG-s-PEI NAs and DTX/IND/PEG-b-PEI NAs were remarkably enhanced (Fig. 5c).
Targeting delivery DTX to tumor for cancer therapy. During the whole treatment, we monitored no abnormal change in the body weight of animals treated (Fig. 6d). However, significant differences on tumor   mice administered with corresponding DTX/IND/PEG-b-PEI NAs and raw DTX (Fig. 6b). DTX concentration in tumors of DTX/IND/PEG-s-PEI treated group was much higher than that of other groups treated with raw DTX and DTX/IND/PEG-b-PEI (Fig. 6c).
The plasma levels of typical biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea (UREA) and creatinine (CREA), which are relevant to liver and kidney functions, showed no significant increase (Fig. 6e,f). Additionally, we did not detect evident injuries or abnormalities in H&E sections of major organs such as heart, liver, spleen, lung and kidney (Fig. S6a), and no significant difference was found in organ index and Hematological parameters (Fig. S6b,c).

Discussion
Cleavable PEGylation is a hot topic in drug delivery system to give vehicle long blood circulation time and efficient phagocytosis by tumor cells. However, low drug loading capacity, complicated materials synthesis, high cost and hardly reproducible manufacturing are limiting its successful translation from bench to bedside, which is also an enormously challenge in the whole novel drug delivery system. In our previous studies, we constructed a facile,  convenient, cost-effective and easily scalable one-pot strategy to assemble various lipophilic therapeutics into nanomedicines, through which highly effective cargo loading and nanoparticles formation can be reached simultaneously. Besides dramatically improving the water solubility, the assembled nanopharmaceuticals show significantly higher bioavailability and better therapeutic activity. This strategy brings the dawn to translate novel drug delivery systems in laboratory from bench to bedside. Therefore, we hypothesize that PEGylation cleavable nanotherapeutics with high drug loading capacity, long blood circulation time and efficient phagocytosis by tumor cells, can be easily constructed by combining cleavable PEGylation with this facile, convenient, cost-effective and easily scalable one-pot self-assembly strategy discovered by our previous studies. In order to validate our hypothesis, we constructed a tumor targeting system of docetaxel based on multiple interactions between indomethacin and PEGylation cleavable PEI (PEG-s-PEI). In this case, IND/PEG-s-PEI nanoassemblies functioned as nanovehicles for DTX, and IND was also employed as COX inhibitor to reverse anorexia induced by docetaxel-based chemotherapy 42 , leading to an IND-DTX combined nanotherapy.
In this conceptual proof study, PEGylation cleavable PEI was firstly synthesized based on pH-sensitive linkage Schiff base and PEI, a commercially available homopolymer that has been widely utilized for both in vitro and in vivo gene delivery [43][44][45] , and also been used as a potent mucosal adjuvant for viral glycoprotein antigens most recently 46 . To determine a most suitable PEG-s-PEIs used in our study, three PEG-s-PEIs with PEG/PEI monomeric molar ratios 2/1 (PEG-s-PEI -1), 4/1 (PEG-s-PEI-2) and 8/1 (PEG-s-PEI-3) were synthesized. PEG-s-PEI -3 was cross-linked to be insoluble, which was firstly abandoned. PEG-s-PEI -1 and PEG-s-PEI-2 were successfully synthesized with well soluble ability in menthol and water, and both showed rapid pH sensitive cleavage of PEGylation from PEG-s-PEIs in acidly environment containing pH 5.5 to pH 6.5 (Fig. S2).
Guided by PEI may interact with small molecules containing carboxyl group to render NAs by multiple noncovalent forces to serve as efficient nanocontainers for other hydrophobic drugs, we found that there were similar interactions between IND and PEG-s-PEI by Autodock programs. Though PEGlyation decreased the electrostatic forces between PEG-s-PEI and IND, pi-pi stacks were supplemented by benzene rings in PEG-s-PEI. So PEG-s-PEI only displayed slightly lower intermolecular energy compared to IND and PEI interaction (Fig. 2c,d), that was notably higher than that of other combinations in IND/PEG-s-PEI system (Fig. 2a). Consistent with the computational results, DTX/IND/PEG-s-PEI-1 and DTX/IND/PEG-s-PEI-2 NAs at various DTX/IND/ PEG-s-PEIs weight ratios were successfully conducted by dialysis against deionized water using methanol as the common solvent that spherical nanostructures with narrowly dispersed size were observed by TEM (Fig. 3a). Conducive to reproducible manufacturing and quality control, the size, ζ-potential and drug loading contents of these NAs could be easily controlled by DTX feeding (Fig. 3b-d). Contributed by PEGylation cleavage of PEG-s-PEIs in acidic conditions (Fig. S2), we monitored the pH-sensitive zeta potential shifting of DTX/IND/ PEG-s-PEIs NAs at pH 5.5 and pH 6.5 (Fig. 4a,b), which imply that exposure of DTX/IND/PEG-s-PEIs NAs to pH 5.5 and pH 6.5 triggered the cleavage of PEGylation, and further led to strong shift of zeta potential of NAs. Importantly, NPs derived from PEI cannot be used in intravenous injection owned to the lethal toxicity of PEI leaded by hemolysis and thrombus rapidly formatted, which greatly limit the clinical transformation of these NPs. The PEGlyation of PEI was proved to improve the biocompatibility of PEI, such as hemolysis ratio (Table S2) and cytotoxicity (Fig. S2c,d). Despite the hemolysis ratio of DTX/IND/PEG-s-PEI-1 was lower than that of DTX/ IND/PEI, it was still in dangerous degree, DTX/IND/PEG-s-PEI-2 showed a safe hemolysis ratio to contact with blood that was contributed by the higher PEG/PEI monomeric molar ratio (Table S2), which was employed in followed in vitro and in vivo experiments.
We next verified that the deshielding of PEGylation at pH 6.5 would facilitate the cellular internalization of NAs and increase DTX accumulation in tumor cells, leading to superior anti-tumor activity (Figs 4 and S4,5). DTX/IND/PEG-s-PEI-2 NAs showed significantly lower anti-tumor activity than DTX/IND/PEG-b-PEI NAs in normal medium, but comparable anti-tumor activity to DTX/IND/PEG-b-PEI NAs in acidy medium. As we know, acidic extracellular microenvironment was proved to decrease the chemo-sensitivity of tumor cells 47,48 . We found the cytotoxicity of raw DTX, DTX/IND/PEG-b-PEI and DTX/IND/PEI were significantly decreased in acidy medium. In this case, the anti-tumor activities of DTX/IND/PEG-s-PEI-2 NAs at low DTX concentrations in acidy medium was inhibited with no improvement than normal medium, even though the DTX accumulation in tumor cells was significantly increased. However, the anti-tumor activities of DTX/IND/PEG-s-PEI-2 NAs at high concentrations were higher in acidy medium than normal, and significant difference between IC 50 at pH 6.5 and 7.4 was still observed for DTX/IND/PEG-s-PEI-2 NAs, which was contributed by the more DTX accumulated in tumor cells that offset the impact of acidic environment on chemo-sensitivity.
To interrogate the drug delivery capacity of DTX NAs, both in vitro and in vivo evaluations were performed. Computational results showed that IND was "embraced" by phenyl group and imine chains of PEG-s-PEI, when PEGlyation was cleaved from PEG-s-PEI-2 in acidy medium, the pi-pi stacking between phenyl group of PEG-s-PEI-2 and IND was abolished that one part of IND molecular would be exposed, and the interaction force between IND and PEG-s-PEI faced the risk of reducing. Especially when PEG-s-PEI degraded fast in pH 5.0, there was no time for IND to reassemble with retained PEI chains, leading the drug release from NAs highly accelerated. Related to the different degradation speeds of PEG-s-PEI under various pH conditions (Fig. S2), DTX/IND/PEG-s-PEI NAs exhibited an excellent pH-triggered release behavior at pH 5.0 stimulating pH in lysosomes, while it was slightly faster DTX release at pH 6.5 stimulating pH e in tumor microenvironment than pH 7.4 (Fig. 5a). Apparently, DTX/IND/PEG-s-PEI NAs exhibited more excellent pH-triggered release and PEGylation cleavage behaviors at pH 5.0, and only pH-triggered PEGylation cleavage at pH 6.5 with little change at drug release behavior than pH 7.0. These behaviors provided the basis for NAs to firstly PEGylation deshielded in tumor microenvironment and then be delivered into tumor cells. Benefit of PEGylation, DTX/IND/PEG-s-PEI NAs and DTX/IND/PEG-b-PEI NAs showed obvious long circulation behaviors at 10 mg/kg of DTX than raw DTX in vivo (Fig. 5b), which resulted in remarkably enhanced t 1/2 (Fig. 5c).
Studies on in vivo antitumor activities of various formulations were conducted that mice received DTX/IND/ PEG-s-PEI NAs showed significantly enhanced anti-tumor activity compared with raw DTX and DTX/IND/ PEG-b-PEI NAs (Fig. 6a-c). Meanwhile, the anorexia induced by docetaxel-based chemotherapy was controlled that body weights of mice received IND-DTX combined nanotherapies were higher than mice received raw DTX (Fig. 6d). These results demonstrated the superior efficacy of newly assembled carriers for targeted delivery of hydrophobic drugs for tumor therapy based on tumor microenvironment. Importantly, this system performed a good safety that no significant toxicity of DTX/IND/PEG-s-PEI NAs was observed in 14 days treatment (Figs 6d-f and S6a-c), and PEG-s-PEI seems to be safe in cytotoxicity even at dose of 20 µg/mL (Fig. S2c,d) both in pH 6.5 and pH 7.4 mediums, which is much higher than that to be used in in vivo evaluations and therapy. However, comprehensive and long term toxicity evaluation is necessary to further study the potential toxicity of accumulated free PEI derived from PEG-s-PEI in vivo.
In summary, a pH-sensitive PEGylation cleavable PEGylated PEI (PEG-s-PEI) was successfully synthesized, and DTX was highly efficiently packaged into NAs driven by multiple noncovalent interactions-mediated host-guest assembly of IND and PEG-s-PEI. These NAs, with desirable redispersibility and scalability, exhibited excellent pH-sensitivity PEGylation cleavage performance between pH e of tumor microenvironment and normal tissues. By being PEGylation selectively cleaved under the acidy conditions in tumor microenvironment, these NAs exhibited a significantly charge shift and highly efficient phagocytosis by tumor cells, while long blood circulation time was retained by PEG chains stretching in circulation. This smart pH-sensitive PEGylation cleavage provided an efficient strategy to target tumor microenvironment, in turn afforded superior therapeutic outcome in anti-tumor activity. Compared with other strategy to target tumor site, this delivery strategy performed several advantages containing prolonged circulation, accumulation in tumors, highly efficient cellular internalization and rapid intracellular drug release. More importantly, this delivery system could be facile fabricated by one-pot assembly and universal drugs applicable, which is providing a new strategy to fabricate the next generation of drug delivery systems and will achieve better therapeutic effects in cancer treatment. Synthesis of PEGylated PEIs. The synthesis of PEGylated PEI linked by Schiff base (PEG-s-PEI) was achieved via a two-step process (Fig. S1a). mPEG-CHO was firstly synthesized by mPEG (MW = 2000) and 4-Formylbenzoic. Then the reaction was carried at various PEG/PEI monomeric molar ratios for PEG-s-PEI-1 (2/1), PEG-s-PEI-2 (4/1),and PEG-s-PEI-3 (8/1). After reaction, the solution was concentrated, the resulting mixture was transferred to a dialysis membrane (MWCO: 3,500) against distilled water for 2 days. And PEG-s-PEIs were freeze dried from dialyzed solution. PEGylated PEI linked by amide linkage (PEG-b-PEI) was synthesized as previous report by mPEG-NHS (MW = 2000) and PEI at monomeric molar ratio of 4/1 49 . The number of PEG linked at PEI was calculated from 1 H NMR spectra by the integral intensities of the signals at 3.38 ppm (CH 3 O-) and signals at 2.3-3.3 ppm (-CH 2 CH 2 -NH-). PEG-s-PEIs were firstly solved in water, and separated on the cation-exchange column, the amounts of unreacted PEG were pooled. Then, the amount of PEG was quantified by measuring the absorbance at 340 nm, the main absorbance of the released aldehyde.

Materials
Hydrolysis Assay. The PEG-s-PEIs were adjusted to pH 5.5, 6.0, 6.5 and 7.4 by the addition of acetic acid to a final acetate concentration of 0.2 M total volume 2 mL and was incubated for 5 h at 37 °C. The reaction was applied to and separated on the cation-exchange column, the unbound PEG fractions were pooled, and the amount of PEG was quantified at different time points by measuring the absorbance at 340 nm.
Computational Studies. Polymers with 50 units were built in three-dimensional (3D) coordinates using MOE's (Molecular Operating Environment software package, Chemical Computing Group, Canada) builder tool. And 5 units was employed to build as small version of polymer to stimulate self-inter-molecular interaction in docking process. The 3D structures of IND, polymer chains, small version of polymer and water molecular were preoptimized before running simulations using the all atom MMFF94x force field with no constraints. For all docking calculations, the size of the grids was set at 126 × 126 × 126 Å using grid spaces at 0.375 was probed to find the most favorable drug-polymer complex geometry (interactions), Lamarckian Genetic Algorithm (LGA) built in Autodock4.2 was probed with 100 docking runs, and LGA searching algorithms the number of energy evaluation was set to 25,000,000 while the population size was set to 150. The other docking parameters were set to the default values. The docking results were analysed by Autodock tools (ADT 1.56) and MOE, while interactions bewteen polymer/repeat unit was estimated by small version polymer docked to polymer and caluated by the unit with highest interaction energy.
Fabrication of Nanoassemblies. NAs were prepared by a dialysis procedure. Briefly, DTX, IND and polymer (PEG-s-PEIs, PEG-b-PEI or PEI) at different feeding ratios were dissolved in methanol. The polymer concentration was 10 mg/mL. Thus obtained solution was dialyzed against deionized water at 25 °C. The outer aqueous solution was exchanged every 2 h. After 24 h of dialysis, DTX/IND/polymer NAs were attained and collected for analysis without further treatment. The drug content in the lyophilized samples was quantified by high performance liquid chromatography (HPLC). The drug loading content and entrapment efficiency were calculated according to the following equations: The weight of drug in nanoassemblies (mg) The weight of nanoassemblies (mg) 100% (1) In vitro Hemolysis Assay. Various NAs were mixed with whole blood and incubated for 45 minutes at 37 °C.
The cells are centrifuged and the absorbance of the supernatant, which includes plasma and lysed erythrocytes, is measured. Percent lysis is calculated from a standard curve of lysed erythrocytes.
In vitro pH triggered potential shift and particle size changes. Particle size and ζ-potential of NAs incubated in various buffers were measured at determined time point. Measurements at pH 7.4 were performed in PBS (0.01 M, pH 7.4), while for pH 5.5 and pH 6.5 measurements, pH values were adjusted by the addition of acetic acid to a final acetate concentration of 0.2 M total volume 2 mL.
Intracellular uptake of NAs. B16F10 and HepG2 cells were separately seeded in a 24-well plate with a density of 1.0 × 10 5 cells per well in 500 μL growth medium at pH 6.5 or 7.4 adjusted by the addition of acetic acid. Cells were incubated at 37 °C with 5% CO 2 for 24 h. Then the culture medium was replaced by 500 μL of fresh medium (pH 6.5 or pH 7.4) containing DTX/IND/PEG-s-PEI NAs (fabricated based on a formulation with DTX/ IND/PEG-s-PEI weight ratio of 10:10:10), respectively. After incubation for various times, cells were washed with PBS and lysed. The drug content was measured by HPLC, while the content of total proteins was quantified by a BCA method. The content of cellular drug was normalized to the protein content.
In vitro release study. For in vitro release tests, 0.5 mL of freshly prepared NAs was placed into dialysis tubing (MWCO: 3500 Da), which was immerged into 40 mL of PBS (0.01 M) at pH 5.5, 6.5 or 7.4. At predetermined time intervals, 4.0 mL of release medium was withdrawn, and the same volume of fresh PBS was supplemented. DTX concentration in the release buffer was quantified by HPLC.
In vivo pharmacokinetic study. All  Assessment of antitumor activity of DTX-containing assemblies. Tumor xenografts were generated by inoculating B16F10 murine melanoma cells into the right limb armpits of athymic nude mice. After 10 days, when the average volume of the xenograft tumors reached 50 mm 3 , the mice were randomly divided into 4 groups (n = 6), for which saline and various nanoassembiles formulations of DTX/IND/PEG-s-PEI,DTX/ IND/PEG-b-PEI, and pristine DTX were administered (i.v) every four days at the DTX dose of 10 mg/kg, respectively. After 15 days, all mice were sacrificed, and the collected tumors were weighed, washed with cold saline, dried on filter paper, weighed and cut into small pieces. The amount of DTX in the tumor was determined by HPLC as previously reported. Blood samples and main organs were also collected for further analysis.
Measurements. 1 H NMR spectra were recorded on a Varian INOVA-400 spectrometer operating at 400 MHz. FT-IR spectra were acquired on a Perkin-Elmer FT-IR spectrometer (100 S). Gel permeation chromatography (GPC) measurement was carried out using a Waters model 440, equipped with a Wyatt Optilab Refractive Index detector. Dynamic light scattering (DLS) and ζ-potential measurements were performed on a Malvern Zetasizer Nano ZS instrument. The freshly prepared samples were diluted according to their scattering intensities for size determination. Unless stated otherwise, measurements were implemented at 25 °C. Transmission electron microscopy (TEM) observation was carried out on a TECNAI-10 microscope (Philips, Netherland) operating at an acceleration voltage of 80 kV.