Nanoparticles of pH-responsive, PEG-Doxorubicin Conjugates

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Nanoparticles of pH-responsive, PEG-Doxorubicin Conjugates: Interaction with an in vitro Model of Lung Adenocarcinoma and their Direct Formulation in Propellant-based Portable Inhalers KSV Krishna Rao, Qian Zhong, Elizabeth R Bielski, and Sandro R. P. da Rocha Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00584 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 8, 2017

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Molecular Pharmaceutics

Nanoparticles of pH-responsive, PEG-Doxorubicin Conjugates: Interaction with an in vitro Model of Lung Adenocarcinoma and their Direct Formulation in Propellant-based Portable Inhalers

K.S.V. Krishna Rao1,2, Qian Zhong2,3†, Elizabeth R. Bielski2,3, Sandro R. P. da Rocha2,3*

1

Polymer Biomaterial Design and Synthesis Laboratory, Department of Chemistry, Yogi Vemana University, Kadapa 516003, Andhra Pradesh, India.

2

Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202, United States. 3

Pharmaceutics and Chemical and Life Science Engineering, Virginia Commonwealth University, 410 N 12th Street, Richmond, Virginia 23298, United States

*To whom correspondence should be addressed: [email protected]; Tel: +1 (804) 828-0985 † Current address: Department of Radiology, Stanford University, Palo Alto, California 94304, United States.

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Abstract Pulmonary administration of polymer drug conjugates is of great potential clinical significance for treating lung cancer as such regimen significantly increases local drug concentrations while decreases systemic and local side effects. In this work, we demonstrate that nanoparticles prepared with methoxypoly(ethylene glycol) (mPEG)-doxorubicin (DOX) conjugates (mPEGDOX) that have a pH-sensitive imine bond (Schiff base) can at the same time work as efficient carriers for DOX to kill cancer cells, and also as a strategy to directly formulate nanoparticles in propellant-based inhalers. Nanoparticles prepared by precipitation in water had a diameter in the range between 100-120 nm. We investigated the effects of molecular weight (MW) of mPEG (1K, 2K, and 5K Da) on the in vitro release kinetics, cellular internalization and cytotoxicity on in vitro model of lung adenocarcinoma and aerosol characters. It is observed that the DOX released from mPEG-DOX nanoparticles was significantly accelerated in acidic environment pH 5.5 (endosomal/lysosomal pH) in comparison with pH 7.4 (physiological pH), as designed. Release of DOX from mPEG1K-DOX nanoparticles was significantly greater than those from mPEG2K and mPEG5K counterparts. In vitro cytotoxicity of nanoparticles, followed the sequence of mPEG1K-DOX > free DOX > mPEG2K-DOX >> mPEG5K-DOX, a trend closely following their rate and extent of cellular internalization. mPEG-DOX nanoparticles with mPEG1K and mPEG2K were directly dispersed in hydrofluoroalkane (HFA), while a trace of ethanol was required to disperse mPEG5K-DOX nanoparticles in HFA. These pMDI formulations with high physical stability in HFAs display superior aerosol characteristics conducive to deep lung deposition. The fine particle fractions of these formulations ranged from 40% to 60%, higher than those of commercial products. Such formulations prepared from nanoparticles of pH-sensitive PEG-drug conjugates may also be envisioned to be extended to 2 ACS Paragon Plus Environment

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formulate other hydrophobic drugs for local delivery with propellant-based inhalers to other pulmonary disorders, thus broadening the impact of the proposed strategy. Keywords: pulmonary drug delivery, pressurized metered-dose inhalers, poly(ethylene glycol), doxorubicin, , Schiff base, pH sensitive conjugates, lung cancer.

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1. Introduction Lung cancer is the most common type of cancer among both men and women worldwide, and is the leading cause of cancer death (ca. 25%), with an estimated 160,000 deaths per year in the United States alone.1, 2 At present, common treatments for lung cancer include surgery (when feasible), radiation, immunotherapy, and chemotherapy, or a combination.3, 4 In spite of its broad applicability, a major challenge with the clinical use of chemotherapy that is yet to be addressed is the very low dose of chemotherapeutic agent (CTA) that reaches the lung tumor upon systemic administration, and their dose-limiting toxicity.5-8 In this context, the local administration of CTAs to the lungs holds great promise in the treatment of lung cancers.

In vivo pre-clinical studies have demonstrated that pulmonary

delivery of CTA can reduce tumor burden, while maintaining a low toxicity profile in healthy tissues due to improved systemic biodistribution.9-11 For example, accumulation of CTAs (e.g. doxorubicin, fluorouracil) in the cardiac tissue, which is related to the dose limiting toxicity characteristic of several potent chemotherapeutics when administered i.v., may be reduced upon pulmonary administration.9, 12 The use of nanotechnology in combination with the oral inhalation route may further improve the therapeutic benefits of CTA regimens in the treatment of lung cancers. Nanocarriers can be used to control both spatial and temporal release of the CTA,13 and also to target tumor cells and cellular organelles.14, 15 Moreover, recent studies have also shown that nanocarriers can enhance penetration of CTA in the solid tumor,16 improve biodistribution and passive targeting of relevant tissues in patients with lung cancer, as for example the lymph nodes,17,

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thus

yielding enhanced efficacy and at the same reduced toxicity of the CTAs.

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Various polymeric drug nanocarriers have been developed to fulfill this objective, including drug-encapsulated nanoparticles/microparticles such as poly(lactide-co-glycolide) (PLGA),19 and stimuli-responsive polymer-drug conjugates including polyethylene glycol,20 dendritic polylysine,11 poly(ethylene imine),10 polyamidoamine,9 and polyester dendrimers.21 One important characteristic in the design of such nanocarriers is their translational potential, particularly when the lung is the tissue of interest. Unique to the pulmonary route of administration is the extremely limited range of non-generally recognized as safe (GRAS) excipients that can be found in FDA-approved formulations.22 Polyethylene glycol (PEG) is one such material, which has been listed in an oral inhalation product that is currently in the market in the form of a pressurized metered dose inhaler (pMDI) – PEG is used in Intal® (PEG 600Da) (discontinued) and Symbicort® (PEG 1000Da) as excipient to improve drug delivery efficiency and dose uniformity.23 PEG can be utilized to modify the surface properties of nanocarriers,24,

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or PEG can be used as the

nanocarrier itself as in PEG-drug conjugates.26 PEGylation of carrier surfaces has been shown to confer various advantages including reduced toxicity,15,

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prolonged residence times in

circulation,21 high serum stability and improved biodistribution.28, 29 Based on the potential opportunities in delivering CTAs locally to the lungs with nanocarrier-based strategies, and limitations in the range of excipients in FDA-approved oral inhalation formulations, the goal of this work was to prepare nanocarriers based on PEG-CTA conjugates with pH sensitive bonds, and to formulate such nanocarriers in propellant-based, portable oral inhalation devices for local lung delivery. Doxorubicin (DOX) was selected as CTA for this study given its high potency and broad use in cancer chemotherapy in the clinic,30 as well as in clinical trials for the treatment of lung cancer via pulmonary route.31, 32 We were also


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motivated by recent pre-clinical success of dendrimer-DOX conjugates published by our group and others,9-11 where a significant reduction in tumor burden in lung metastasis in vivo was observed with DOX, even for models where DOX would not have been the first choice. DOX was conjugated to mPEG via an acid-labile imine bond for enhanced stability under extracellular physiological conditions and intracellular drug release. The reaction conditions were optimized, and conjugation of DOX to mPEG 1KDa, 2KDa and 5KDa was assessed by 1HNMR and FTIR. Polymeric nanoparticles with the mPEG-DOX conjugates were prepared by precipitation in water, and their size and morphology characterized by light scattering and TEM. The cumulative release of DOX was measured as a function of pH and mPEG MW. The rate and extent of cellular internalization was assessed by flow cytometry in an in vitro model of lung adenocarcinoma (A549 cells), also as a function of mPEG MW, and those results used to understand the ability of such the nanoparticles to kill the cancer cells. The nanoparticles were formulated (dispersed) in hydrofluoroalkane (HFA)-based pMDIs, and their aerosol characteristics (lung deposition) assessed using an Andersen Cascade Impactor. Such study is relevant given the potential in using such nanocarriers for DOX in the treatment of lung cancers, and the enhanced distribution of such carriers upon local pulmonary administration. Such strategy can be potentially used to deliver other hydrophobic drugs to the lungs using pMDIs. Nanoparticles have also shown to be able to be formulated in dry powder inhalers (DPIs),33, 34 and thus further extending the relevance of this work. 2. Materials and methods 2.1. Materials. Doxorubicin hydrochloride (DOX·HCl) was received from LC laboratories (Woburn, MA, USA). Anhydrous methyl alcohol (MeOH), dichloromethane (DCM), diethyl


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ether, glacial acetic acid, 4-formylbenzoic acid (FBA), and 4-(dimethylamino)pyridine (DMAP) were purchased from Alfa Aesar (Ward Hill, MA, USA). Methoxy-poly(ethylene glycol) of molecular weight 1000 Da, 2000 Da, and 5000 Da (mPEG1K, mPEG2K and mPEG5K) and dicyclohexylcarbodiimide (DCC) were received from Aldrich Sigma (St Louis, MO, USA). Deuterated dimethylsulfoxide (DMSO-d6) was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). The propellant 1,1,1,2,3,3,3-heptafluoropropane (HFA-227) was a gift from Dupont (Wilmington, DE, USA). All chemicals were used as received. Ultrapure deionized water (DI H2O; Resistivity=18.0-18.2 mΩ) from Thermo Fisher Scientific (Waltham, MA, USA) was used in cell experiments. Sterile syringe filters (0.22 µm), tissue culture treated 96-well plates and 24-well plate were purchased from VWR Internationals (Radnor, PA, USA). Human lung alveolar adenocarcinoma epithelial cell line (A549) was purchased from American Type Culture Collection (Manassas, VA, USA). The cell toxicity assay compound 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin (10,000 U/ml)-streptomycin (10,000 µg/ml) (Pen-Strep), trypsin– EDTA were purchased from Life Technologies (Grand Island, NY, USA). Fetal Bovine Serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA, USA). Spectra®Por dialysis membrane (MWCO=1000Da) was purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA) 2.2. Preparation of mPEG-formylbenzoic acid (mPEG-C6H5-CHO).35 mPEG2K (1.28 g, 0.64 mmol), and FBA (0.11 g, 0.73 mmol) were dissolved in 20 mL anhydrous DCM, and the resulting solution was stirred in an ice bath. Then DCC (0.21 g, 1 mmol) and DMAP (0.12 g, 0.1 mmol) were added to the reaction solution. The solution was kept under stirring overnight. After removing dicyclohexylurea (DCU) by filtration, the filtrate was concentrated using a rotary


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evaporator and it was precipitated in dry diethyl ether and dried at 40 oC. mPEG1Kformylbenzoic acid (mPEG1K-C6H5-CHO) and mPEG5K-formylbenzoic acid (mPEG5K-C6H5CHO) were prepared by same method. Yields: 83-87 %. 2.3. Neutralization of DOX·HCl with NaOH. DOX·HCl aqueous solution (25 mL at 2 mg/mL) was carefully neutralized using 0.1 N NaOH aqueous solution. The neutralized doxorubicin (DOX) was extracted with ACS grade DCM (50 mL×5) and the resulting DCM solution was dried under reduced pressure to give a brown red product. 2.4. Synthesis of acid-labile mPEG-DOX conjugates. mPEG-DOX Schiff base was prepared by taking 19.56 mg mPEG2K-C6H5-CHO into 5 mL anhydrous MeOH and then adding catalytic amount of acetic acid with vigorous stirring. Anhydrous MeOH (2 mL) containing 5 mg DOX was added dropwise to the above solution. The reaction mixture was purged with nitrogen gas for 5 min, the reaction was refluxed for 12 h and then the solvent was removed under reduced pressure to obtain a reddish white product. The product was purified by dissolution in MeOH and precipitation with cold anhydrous diethyl ether. The precipitate was dried thoroughly. mPEG1KDOX and mPEG5K-DOX were prepared by same method. 2.5. Characterization of acid-labile mPEG-DOX conjugates. mPEG-DOX conjugates were characterized by attenuated total reflection Fourier transform infrared (FTIR-ATR) spectroscopy using a NICOLET 6700 spectrometer (Thermo Scientific). The spectra were scanned from 500 to 4000 cm-1. Spectra are provided in Figure S1 in Supporting Information. Proton nuclear magnetic resonance (1H NMR) spectra of mPEG, mPEG-C6H5-CHO and mPEG-DOX were recorded on a Mercury 400 MHz spectrometer (Agilent Technologies). The solvent peak of


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DMSO-d6 at 2.487 ppm was used as reference peak. Chemical shifts are expressed in ppm and the values are given in the δ scale. 2.6. Preparation and characterization of the nanoparticles. mPEG-DOX nanoparticles were prepared by a simple precipitation technique. The mPEG-DOX conjugate (20 mg) was dissolved in 1.5 mL of anhydrous methanol and added dropwise into 20 mL of phosphate buffer solution at 25 oC. The resulting mPEG-DOX nanoparticles were collected through centrifugation at 10,000 rpm with Sorvall Legend X1 centrifuge (Thermo Scientific). The size and morphology of mPEG-DOX nanoparticles were measured with transmission electron microscope (TEM, FEITecnai G2 20 S-TWIN) operated at an accelerating voltage at 200 kV. The hydrodynamic diameters (HD) and zeta potential (ξ) of the mPEG-DOX nanoparticles were measured using Zetasizer Nano ZS (Malvern Instruments Ltd) in phosphate buffer saline (PBS; 1X, pH 7.4) at 25 o

C. The sample concentration of the mPEG-DOX nanoparticles was 0.1 g/mL (n=10 per sample).

2.7. In vitro release of DOX from mPEG-DOX nanoparticles. In vitro release of DOX from the nanoparticles was studied at 37 oC by using the Labquake™ Shaker Rotisserie (Thermo Scientific) set to 100 rpm at pH 7.4 and pH 5.5, which mimic extracellular/physiological pH and endosomal/lysosomal pH, respectively. The release at pH 7.4 was performed at sink condition as follows: A 2 mL PBS (pH 7.4, 1X) of free DOX or mPEG-DOX nanoparticles (9.1 mmol DOX) were introduced into a Spectra®Por dialysis bag (MWCO=1000 Da) and the dialysis bag was immersed in 30 mL PBS, which is used as the release medium. At pre-determined time intervals, 200 µL of the release medium was removed and measured for UV absorption at λmax=490 nm with Multiscan GO plate reader spectrophotometer (Thermo Scientific). The release medium was then returned to the reservoir after UV measurement. The cumulative release of DOX was quantified with a pre-established calibration curve. The calibration curve of DOX absorption at


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490 nm with respect to mass concentration of DOX was obtained by using UV spectrometric method (Figure S2). The release fraction of DOX from mPEG-DOX-based nanoparticles was calculated by using following equation:

Cumulative release % =

∞

× 100%

(Equation 1)

where Mt is the amount of DOX released from the mPEG-DOX nanoparticles at time t and M∞ is the amount of DOX pre-loaded in mPEG-DOX particles. The release experiment was performed in triplicate. It is noted that the recovery rate (measurable percentage) of DOX in the release experiment is about 90-95% due to the binding to cellulose dialysis tubing and other losses (only 90-95% of initial DOX dose can be measured). A similar procedure was applied to the DOX release at pH 5.5 with an exception of the replacement of 1X pH 7.4 PBS with 1X pH 5.5 citrate buffer. We also modeled the release kinetics of DOX from the mPEG-DOX nanoparticles. The linear relationship observed at initial times (1-8 h) can be represented as:

Mt = kt n M∞

(Equation 2)

where, Mt/M∞ represents the cumulative drug release percentage at time t, k is a constant and is characteristic of the drug-polymer system, and n is an empirical parameter characterizing the release mechanism. 2.8. Culture of A549 Cells. A549 cells (passages 5 to 10), an adenocarcinoma human alveolar epithelial cell line, were plated in Corning™ cell culture treated 75 cm2 cell culture flasks (canted neck and vented cap) at a density of 104 cells/mL, and cultured in DMEM supplemented


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with 10% FBS (v/v) and 1% Pen-Strep (v/v). The cells were grown in thermally controlled Thermo Scientific™ CO2 incubator (Thermo Fisher Scientific) with constant purging of 5% CO2 at 37 oC. The medium was exchanged every two days, and the cells were split when they reached ca. 80% confluence. 2.9. Cytotoxicity of acid-labile mPEG-DOX nanoparticles. The ability of DOX, and of the nanoparticles prepared with mPEG1K-DOX, mPEG2K-DOX and mPEG5K-DOX conjugates to kill A549 cells was assessed using the MTT assay. A series of concentrations of free DOX or mPEG-DOX nanoparticles in DMEM were sterilized through 0.22 µm sterile syringe filter (VWR Internationals). Approximately 5000 cells/well (n=6 per concentration) were seeded in tissue culture treated 96-well plate with DMEM. The medium was removed after 24 h, and 100 µL of the sample-laden DMEM (10% FBS, 1% Pen-Step) was pulsed onto each well. The cells were incubated with samples (free DOX or mPEG-DOX) for 48 h. The sample-laden medium was then removed from each well and then washed with PBS (1X, pH 7.4) twice. 100 µL of fresh PBS (pH 7.4, 1X) and 10 µL of MTT PBS solution (5 mg/mL) were added to each well. After 4 h incubation (37 oC, 5% CO2), 75 µL of medium was removed, and 60 µL DMSO was added into each well to dissolve the formazan crystals. The cells were then allowed to sit in the incubator (37 oC, 5% CO2) for another 2 h. Finally, the absorbance of each well was recorded at 540 nm using Multiskan GO Microplate Spectrophotometer (Thermo Scientific). The cell viability was calculated with the equation: Cell viability (%) = ( − )⁄( − ) × 100 %

(Equation 2)

where As, Anc, and Apc are absorbance at 540 nm of sample, negative control (100% viable cells), and positive control (100% dead cells). Cells only incubated in medium in the absence of DOX


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represent the negative control group; wells filled in with only medium (no cells) represent the positive control group. 2.10. Cellular uptake of mPEG-DOX nanoparticles. Cellular uptake studies of mPEG-DOX nanoparticles were performed on A549 cells using Invitrogen™ Attune™ Flow Cytometer (Life Technologies), with free DOX being used as control. A549 cells were seeded at a density of 3×105 cells per well in a 24-well plate using DMEM supplemented with 10% FBS and 1% PenStrep, and then allowed to sit for 24 h at the condition of 37 oC and 5% CO2. Prior to incubating A549 cells with mPEG-DOX nanoparticles (DOX equivalent: 1 µM), the cells were incubated with 500 µL blank HBSS (1X, pH 7.4) for 30 min and then 500 µL HBSS of DOX or mPEGDOX was added after removal of blank HBSS. After completion of incubation at 0.25h, 0.5h, 1h, 1.5h, 2h, 3h, 4h, and 5h, the cells of each well were washed with 0.5 mL of 1X HBSS and followed by trypsinization (100 µL of trypsin–EDTA for 5 min). The trypsinized cells were incubated with 0.1 ml 0.2% trypan blue PBS for 5 min to quench extracellular fluorescence of DOX.7 The cells were then centrifuged at 350 g for 5 min, and then the pellet was re-suspended in 1 mL of 1X HBSS (4oC). The extent of DOX taken up by the A549 cells was determined with flow cytometry. For statistical significance, at least 10,000 events were recorded and data were analysed with Attune® Cytometric Software (Life Technologies). The effect of MW of mPEG on the internalization of nanoparticles prepared with the mPEG-DOX conjugates was evaluated. The mean fluorescence intensity (MFI) was plotted as a function of time. The initial rate of internalization is defined by linearly fitting the (MFI, time) at 0h, 0.25h, and 0.5 h. The unit of initial rate of internalization is absorbance unit/hour (a.u.h-1). 2.11. Preparation of the mPEG-DOX nanoparticle formulations in pMDIs and their aerosol performance. A homogeneous dispersion of mPEG-DOX nanoparticles in propellant-based


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(HFA-227) pMDI was prepared. Briefly, mPEG-DOX nanoparticles (1 mg DOX/mL HFA-227) were added to a pressure proof glass vial (West Pharmaceutical Services) and these were crimped manually with a 63 µL metering continuous valve (Bespak). Subsequently, 2 mL HFA227 was added manually to the sealed glass vial using a syringe pump (HiP 50-6-15) and a home-made pressure filler. The sample and propellant mixture was vortexed for 1 min and sonicated for 30 min in VWR P250D low energy sonication bath (VWR Internationals) at 180W and 5 oC. The in vitro aerosol performance of mPEG-DOX nanoparticle aerosol formulations was determined using an Andersen Cascade Impactor (ACI) equipped with a USP induction port (CroPharm, Inc). The ACI was operated with the specifications as per our earlier study,36 including flow rate: 28.3 L/min, temperature: 25 oC and relative humidity: 75%. The prepared pMDI formulations were fired for actuation of 5 shots to waste, followed by 10 shots into the ACI setup with a 10 s interval between each shot. USP requires no less than 5 s between two consecutive actuations, which allows the particles to reach the different stages and allows the system to locally return to the room temperature as it is cooled upon HFA vaporization.37 The ACI was dissembled for cumulative collection the samples form the actuator, induction port, plates and filter. Each part was rinsed with ethanol carefully and made up to the 10 mL volume. The collected mPEG-DOX samples were measured for UV absorbance with Cary 50 UV-Vis spectrometer (Agilent Technologies) at 490 nm using a pre-determined calibration curve. The aerosol performance of mPEG-DOX nanoparticle formulations were examined by the following parameters

38, 39

: respirable fraction (RF, i.e. mass in Stage 0 to filter divided by the mass in the

actuator + mass in induction port and mass in all other stages of ACI), fine particle fraction (FPF; i.e. mass in Stage 3 to filter divided by the mass in the Induction port to filter), mass median


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aerodynamic diameter (MMAD), geometric standard deviation (GSD) and recovery of mPEGDOX nanoparticles (i.e. collected total dose in ACI divided by the theoretical dose obtained by the dispersed volume fraction and the total volume per actuation). According to USP Chapter,37 the percentage of mass less than the stated aerodynamic diameters is plotted with respect to the aerodynamic diameter cutoff on long probability. The MMAD is the intersection point of the line with the 50% cumulative percent. GSD is calculated with the formula (d84/d16)1/2, where d84 and d16 represent the diameters at which 84% and 16% aerosol by mass (the portion of aerosol with diameters ≤ these values, respectively) are included, respectively. The MMAD and GSD were calculated with an online tool available on http://www.mmadcalculator.com/andersen-impactor-mmad.html. 2.12. Statistical Analysis. All the data were collected from at least three independent trials (n=3), the data was processed and presented as mean±standard deviation (S.D.). GraphPad Prism 5 software was used to perform statistical analysis with one-way analysis of variance (oneANOVA) and means were considered statistically significant if p < 0.05. 3. Results and Discussion 3.1. Synthesis and Characterization of Acid-Labile mPEG-DOX. The pH-sensitive Schiff bases of mPEG-DOX with varying MW of mPEG were synthesized via the formation of imine bond between the aldehyde of mPEG-FBA and the primary amine group of DOX, as schematically illustrated in Scheme 1. This acid sensitive bond was selected so as to promote the intracellular release of the CTA (lysosomes),40 so as to minimize unwanted toxicity of DOX towards healthy tissues when in the extracellular physiological environment.41


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It is important to note here, that in spite of previous reports of PEG-DOX conjugates in the literature, the full characterization of such compounds and reaction optimization conditions have not been fully described until now.42-46 In this work we observe and report the characteristic peaks of imine bonds in NMR and infrared spectra. We also report the optimum ratio of amine to aldehyde, as well as the amount of acidic catalyst, which we observed are essential to yield high purity Schiff base of mPEG and DOX. To improve the reaction efficiency of macromolecular DOX Schiff-base we thereby firstly optimized the reaction with various molar ratios (1:0.5, 1:1 and 1:2) of aldehyde (of mPEG-FBA) to amine (of DOX), and the ratios of catalyst (i.e. acetic acid) to amine of DOX (acetic acid 0.2, 0.4, and 0.6 equivalents to amine; data not shown). Finally, the best yield and purity of Schiff-base mPEG-DOX obtained in the study was that with the molar ratio of amine:acetic acid = 1:0.2 and aldehyde:amine =1:2. mPEG (1KDa, 2KDa and 5KDa) was first reacted with FBA in DCM by condensation in presence of DMAP and DCC – Scheme 1A. The resulting mPEG-FBA was purified by dissolving in DCM and subsequent precipitation in anhydrous diethyl ether. The formation of a new peak (not present in mPEG) at about 1729 cm-1 was assigned mostly to the ester bond (COOR) formed between -OH of mPEG and -COOH of FBA via esterification reaction – see Figure S1. The 1H NMR spectrum also showed aromatic aldehyde proton peak from mPEG-FBA at ca. 10.1 ppm (peak s' in Figure 1). The 1H NMR spectra of mPEG-FBA indicates that the


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esterification reaction efficiency between mPEG and FBA was about 99%, as calculated by comparing the integral of the peaks at 10.1 ppm and 3.4 ppm – see Figure 1.

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The aldehyde group of mPEG-FBA and amino group of DOX further yielded the Schiff base linkage between mPEG and DOX in presence of acetic acid. A singlet at ca. 8.24 ppm in 1H NMR represents the signal for imine proton (peaks in Figure 1) and is thus indicative of Schiffbase formation.40 From integrals of the peaks at 8.24 ppm and 3.4 ppm in the 1H NMR spectra, the reaction efficiency of mPEG-DOX was determined to be about 99.5%. The imine (C=N) bond stretch formed by the reaction between the -CHO of mPEG-FBA and -NH2 from DOX at 1651 cm-1 overlaps with the peak corresponding to the N-H stretching vibrations in DOX (Figure S1) and thus cannot be used to ascertain the formation of the C=N bond. As mass-assisted laser desorption/ionization-time of flight (MALDI-TOF) is not able to accurately determine the molecular weight of mPEG-DOX conjugates due to high instability of imine during the ionization (see brief discussion in Supporting Information S2). The molecular weight of mPEG-DOX conjugates with 1HNMR spectrometry. The results are summarized in Table 1. As the 1H NMR results showed the effective conjugation of DOX to PEG (>99% DOX exist in the conjugated form), the Mn of the conjugates are 1701 Da for mPEG1k-DOX, 2652 Da for mPEG2k-DOX and 5590 Da for mPEG5k-DOX, respectively.


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3.2 Preparation and Characterization of the Nanoparticles Formed with mPEG-DOX Conjugates. 3.2.1. Preparation, Morphology, Size and Surface Characteristics of Nanoparticles. In order to formulate the mPEG-DOX conjugates in pMDIs, and take full advantage of the potential of nanocarriers in optimizing drug delivery of CTA agents,47-49 here we prepared nanoparticles with the mPEG-DOX conjugates. The hydrodynamic diameter (HD), polydispersity index (PDI) and zeta potential (ζ) of the nanoparticles prepared with mPEG-DOX conjugates were measured in 1X PBS using LS. The results are summarized in Table 1 as a function of the molecular weight of mPEG.

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The mean HDs of the NPs prepared with mPEG1K-DOX, mPEG2K-DOX, and mPEG5K-DOX are 104±1 nm, 117±1 nm, and 124±2 nm (mean±s.d.), respectively. PDIs are 0.15, 0.28 and 0.33. These results demonstrate that the HD of the mPEG-DOX nanoparticles is only slightly affected by the MW of mPEG, with an increase in mPEG size leading to increase in NP size. Moreover, all the NPs have a relatively narrow size distribution. The geometric size and morphology of the nanoparticles prepared with mPEG1K-DOX conjugates were assessed by TEM to validate the LS results - Figure S3. TEM micrographs show that the nanoparticles displayed a spherical morphology, with an average diameter of 91±6 nm, in excellent agreement


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with the HD diameter from LS. As expected, the mean zeta potentials of the mPEG-DOX NPs are observed to be close to neutral. Nanoparticles with this size range have been shown in the past to be taken up into cells via endocytic pathways,7, 50, 51 many of which will go through acidification, which is desirable for breakup of the imine bond between DOX and mPEG. Nanoparticles of various polymer with PEG-modified surfaces have been widely utilized in drug delivery,49,

52, 53

and help evade

elimination by the kidney due sizes much larger than the pores sizes of the glomerulus - ca. 6 nm in diameter,54 and also by reducing uptake and elimination by RES.55 However, it is important to note that in the context of the local administration to the lungs via pulmonary delivery route, the concerns are somewhat different than those that arise upon oral or i.v. administration of CTAs. Here we would seek to either avoid uptake of the nanoparticles by resident macrophages in the alveolar region, or to transport through the mucosal barrier so as not to be cleared from the lungs. In both cases, one would expect that the PEGylated surfaces of the NPs in question would also have a positive impact.17, 56 3.2.2. In vitro release of DOX from mPEG-DOX Nanoparticles. In vitro release kinetics of DOX from mPEG-DOX NPs as a function of MW of mPEG and pH are summarized in Figure. 2. Release studies are benchmarked with diffusion of free DOX out of dialysis membrane.

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We selected two different pH environments: pH 5.5 mimics the acidity of subcellular endosomes and lysosomes, while pH 7.4 mimics physiological/extracellular pH. Release studies indicate that the free DOX quickly diffuses out of dialysis tubing in two pHs and the diffusion of free DOX is slightly faster at pH 7.4 (ca. 86% at 8 h) than that at pH 5.5 (ca. 79% at 8 h). The slight difference may be because more protonated DOX at pH 5.5 results in higher binding to cellulose-based dialysis membrane tubing.36 In contrast, conjugated DOX in nanoparticle form demonstrated more sustained release at pH 7.4 when compared to pH 5.5, in agreement with the expectation and literature,43, 57 as well as showed release kinetics dependence on PEG molecular weight. At 12 h, 43% of DOX was released from mPEG1K-DOX nanoparticles when particles were maintained at pH 7.4, while 84% release at pH 5.5. Faster release at lower pHs can be attributed to the establishment of equilibrium between Schiff base (imine bond) and free DOX – pH-triggered release. Moreover, at both pH conditions, the longer the mPEG chain, the slower the DOX release from the NPs. The cumulative release of DOX at 12 h from pristine DOX, mPEG1K-DOX, mPEG2K-DOX and mPEG5K-DOX at pH 5.5 is 87%, 85%, 71%, and 58%, respectively. It seems pristine DOX and mPEG-1K-DOX release patterns are similar, due to the chain length and high solubility of mPEG1K. The faster rate and higher extent of DOX release from mPEG1K-DOX at pH 5.5 compared to other mPEG-DOX nanoparticles may be explained by the following: (1) the longer PEG chain (i.e. more random coiled/entangled polymer chains) reduces the access of protons to terminal imine bond, leading to a slower cleavage of imine bonds; (2) an increase in molecular weight of mPEG is expected to lead to a restriction in free diffusion DOX due to more entangled/randomly coiled chains;20 (3) the internal structure of the nanoparticles at a more macroscopic level (not chain entanglement) may be different depending on mPEG MW,


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and that may lead to different rates of diffusion of DOX once the bond is broken down. In addition, pH plays a key role in influencing mPEG diffusion. That is, compared with high molecular weight, the lower molecular weight PEG diffuses faster at low pH.58 Modeling results show that the drug release mechanism of DOX from mPEG1K-DOX and mPEG2K-DOX nanoparticles, at both pHs, mainly fit into anomalous or non-Fickian diffusion as n values fall between 0.43 < n < 1.00,59 as shown in Table 1. The exception is for the release of mPEG2K-DOX nanoparticle at pH 7.4 from which DOX released is governed by Fickian diffusion (0 < n < 0.43).59 In contrast, DOX is released from mPEG5K-DOX nanoparticles through Fickian diffusion - n = 0.15 at pH 5.5, and n = 0.38 at pH 7.4. Fickian diffusion is a solute transport process in which polymer relaxation time (tr) is much greater than solvent diffusion time (td), while as tr is similar to td drug release turns non-Fickian type.60 The higher MW, the more entangled polymer chains are in the nanoparticles, thus leading to longer polymer relaxation time. It can be therefore expected that mPEG5K-DOX nanoparticle holds down the transport of released DOX in nanoparticle matrix due to slower polymer chain relaxation, compared to PEG1K and 2K counterparts. In summary, the release profiles indicate that DOX release from mPEG-DOX NPs can be controlled by both pH conditions (trigged-release) and further modulated with mPEG molecular weight. DOX release will be relatively slower at extracellular physiological environment such as bloodstream, while DOX will be readily released in the acidic environment of endolysosomes upon cellular internalization, which is the intended effect of the conjugation scheme and also formation of the nanoparticles with the conjugate.


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3.3 Cellular Uptake of Nanoparticles Prepared with mPEG-DOX Conjugates. The cellular internalization of free DOX and the DOX in the mPEG-DOX conjugate nanoparticles by A549 cells was evaluated as a function of time (up to 5 h) by flow cytometry. The kinetics was expressed by a plot of mean fluorescence intensity (MFI) of DOX internalized within the cells as a function of the mPEG MW and time, as shown in Figure 3.

> It was observed that the rate and extent of internalization of nanoparticles prepared with mPEG-DOX conjugates was strongly impacted by the mPEG MW. The higher the mPEG MW, the lower the extent of cellular internalization – this could be observed even at early times. A similar trend has been also reported in other studies, including PEGylated liposomes and PEG nanoparticles.49, 61 Compared to free DOX uptake, only mPEG5K-DOX nanoparticle has lower overall extent of internalization, at least within the time frame of the experiment. We also evaluated the early kinetics of internalization using the initial rate of internalization. The initial rates of internalization of free DOX, and mPEG1K-DOX, mPEG2KDOX and mPEG5K-DOX nanoparticles were determined to be 4560 a.u.h-1, 8075 a.u.h-1, 5957 a.u.h-1and 2720 a.u.h-1, respectively, which was approximately 1.77, 1.30, and 0.60-fold of that of free DOX. The results indicate that the rate of uptake of DOX is fastest when in the form of nanoparticles prepared with mPEG1K-DOX conjugates, and decreases as the MW of mPEG increases.


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It has been reported that free DOX is taken up into cells by passive diffusion, which depends on the concentration gradient and hydrophobicity of DOX.62, 63 Nanoparticles on the other hand, are internalized via fluid phase endocytosis.47 Various endocytic pathways, including macropinocytosis, clathrin-mediated endocytosis, and non-specific, adsorptive endocytosis are involved in the uptake of nanoparticles.64 The preferred endocytic pathways are dictated by size, shape and surface chemistry.36 In combination with the release studies shown in Figure 3, one could hypothesize that the relative fast rate of uptake of DOX from the nanoparticles prepared with the mPEG1K-DOX conjugates may arise due to the fact that there are various strategies by which DOX can be taken up in that case, compared to the passive diffusion mechanism (only) for free DOX (Scheme S1 in Supporting Information). The release profile for mPEG1K-DOX nanoparticle at pH 7.4 (Figure 2B) shows that at 5h ca. 34% of DOX has been released. This free DOX from nanoparticles that have not been internalized can be taken up by passive diffusion, while at the same time nanoparticles loaded with DOX can also gain access to the intracellular milieu through fluid phase endocytosis. The trends observed in terms of decrease in rate and extent of internalization of DOX from nanoparticles containing increasing MW of mPEG can also be at least partially rationalized based on the fact that a slower rate of release of free DOX is observed as mPEG MW increases. This should work in tandem to decrease in DOX cellular internalization along with the decreased particle-surface interaction typically reported as the MW of PEG on the particle surface increases.42, 65 3.4. In vitro Cell Kill and Cellular Uptake of mPEG-DOX Nanoparticles. The efficacy of the nanoparticles prepared with the mPEG-DOX conjugates in killing lung tumor cells was tested on an in vitro model using A549 cells, which are derived from human lung adenocarcinoma. IC50


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(µM) values of free DOX and conjugated DOX (NP prepared with mPEG-DOX conjugates) was calculated by fitting cell viability profiles obtained from MTT assays for the various systems with a non-linear regression Log(inhibitor) vs Response (variable slope). The raw results are summarized in Figure 4 and the IC50 estimated from those results are shown in Table 1.

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It is seen that at 48h, the cytotoxicity of mPEG-DOX nanoparticles against A549 cells increases as the molecular weight of PEG decreases. IC50 values obtained for mPEG1K-DOX, mPEG2K-DOX, and mPEG5K-DOX are 0.50 µM, 3.01 µM and 11.69 µM, respectively. It is interesting to note that free DOX (IC50= 0.9 µM) is slightly less toxic than that of nanoparticles prepared with mPEG1K-DOX, while more toxic than nanoparticles containing mPEG2K and mPEG5K. A potential explanation for the difference in cytotoxicity requires the panoramic consideration of extent of cellular internalization and intracellular release kinetics of DOX from nanoparticles.

Flow

cytometry

results

demonstrated

significantly

enhanced

cellular

internalization of DOX by NPs prepared with mPEG1K-DOX conjugates compared to free DOX as discussed earlier, while much lower internalization of DOX from NPs formed with mPEG5KDOX conjugates, which in good agreement with in vitro cytotoxicity of the corresponding nanoparticles and free DOX. Interestingly, mPEG2K-DOX was 3.4-fold less toxic than free DOX, although the cellular internalization of mPEG2K-DOX was significantly greater than that


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of free DOX at 5h. One potential explanation for such observations is that at 48h, the total uptake (and nuclear colocalization of DOX), can be greater for free DOX than in the mPEG2KDOX conjugate form. Indeed, the difference in overall uptake seems to diminish as a function of time for free DOX and NPs prepared with mPEG2K-DOX, with a statistical but small overall gains at 5h – see Figure 3. The difference in intracellular trafficking pathway of free DOX and conjugated DOX also contributes to their differing cell kill.62 It is reported that free DOX quickly diffuses through the cell bilayer and reach the nuclei, while PEG-conjugated DOX is released after internalization to lysosomes where the imine bonds cleave, followed by the diffusion of released DOX out of lysosomes and subsequent transport to the nuclei.36, 66 The diffusion of DOX out of lysosomes is also a time-consuming process as the internal membrane of lysosomes is only permeable to weak bases, while their cationic forms (major form of weak bases in lysosomes) will convert to DOX for outward diffusion.67 Therefore, PEG-DOX conjugates and free DOX were incubated with A549 cancer cells for 48 h to allow the complete release of conjugated DOX and intercalcation of released DOX with nuclear DNA of cancer cells. In addition, the in vitro release also showed that mPEG1K-DOX nanoparticles can achieve 95% release at acidic conditions at 24 h, while the cumulative release of mPEG2K-DOX and mPEG5K-DOX nanoparticles were only 73% and 67%, respectively. It also needs to be emphasized that the in vivo therapeutic efficacy of anticancer drugs relies on various factors. One crucial aspect relates to their dose in the tumor sites (before it can be internalized). It is well known that PEGylated nanoparticles tend to accumulate in tumor tissues compared to free therapeutics,7, 24 to a large extent due to prolonged residence in blood circulation. Therefore, it remains possible that mPEG2K-DOX and mPEG5K-DOX


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nanoparticles may have greater antitumor potency compared to free DOX (in vivo), although they are less toxic than free drug to A549 cells in vitro, which is a fact commonly observed in polymer-drug conjugates.7, 9, 68 In summary, the cytotoxicity results demonstrate that mPEG-DOX nanoparticles exhibit mPEG size-dependent in vitro cytotoxicity towards A549 cells. That is, higher in vitro potency than pristine DOX when small PEG (1000 Da) is used to form the nanoparticles, while less cytotoxicity is observed when PEG larger than 2000 Da is employed. These effects can be rationalized in terms of cellular uptake and release kinetics of those nanocarriers. 3.5. Formulation and Aerosol Characteristics of mPEG-DOX Nanoparticles in Portable, Propellant-based Inhalers. Another objective of this work was to develop a strategy suitable for the delivery of the nanoparticles containing DOX directly into the lungs. Oral inhalation is widely accepted as an effective non-invasive route for pulmonary administration. We are particularly interested in the development of pMDI formulations due to the many advantages of such portable inhalers, including ease of use and the fact that they are the least expensive oral inhalation devices in the market today.69, 70 It is important to note that in spite of relatively small doses that have been demonstrated so far for pMDI formulations compared to other devices (per actuation), we see many potential opportunities even at lower dosages in terms of preventive and (neo- and concurrent-) adjuvant therapies as the local deposition efficiency is much higher compared to i.v. and requirements may be further reduced when combining with nanotechnologies as discussed here. There are many opportunities in developing formulations where higher dosages as well, the limit of which has not been actively sought for the various nanocarriers, including the one being discussed here.


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However, there are several other challenges in formulating nanocarriers for pulmonary drug delivery, including achieving an appropriate aerosol size, which lies in the micro range and not nano range.71 In our previous work, we have reported the formulation of polymeric nanoparticles in the hydrofluoroalkane (HFA) propellants accepted by the FDA for pMDIs that can achieve good lung deposition by developing particle engineering strategies, including incapsulation in biodegradable shells that provide for physical stability for the otherwise nondispersible nanoparticles, and appropriate aerodynamic size.21,

33, 36, 38

The engineering of

micron-sized particles also has another important function, which is to have an appropriate aerodynamic size so as to produce aerosols with sizes conducive to deep lung deposition – which is optimum at around 0.5-5.0 µm.36 We have also recently shown that PEGyaltion can significantly improve the solubility of HFA-phobic polymers (as for example dendrimer nanocarriers) in HFA propellants, by forming pseudo-solution formulations that aerosolize in micron-size loose aggregates from phase separated domains.36 Those formulations were shown to have aerosols with excellent characteristics for deep lung deposition.36 In this work, we show that the engineering of NPs with high density of surface PEG also works to impart steric stability to the nanoparticles directly in HFA - without the need of the extra shell. We also show that such nanoparticle formulation is capable of producing aerosols that are also conducive to deep lung deposition in spite of the nanoscale diameter of the particles. The physical stability of the NP formulations was assessed by visually monitoring the dispersion of the NPs prepared with the mPEG-DOX conjugates in HFA227 as a function of time after sonication.21 The qualitative results are summarized in Figure 5.


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>

We observed that the mPEG1K-DOX and mPEG2K-DOX nanoparticles were easily and well-dispersed in propellant - at least within observation time (5 h), while NPs prepared with mPEG5K-DOX conjugates quickly sedimented in HFA227, even at early times. Our previous ab initio calculations and chemical force microscopy results have revealed that the ether oxygen atom (i.e., -CH2CH2O-, in this case of PEG) can strongly interact with the dipole of the HFA molecule.72 However, self-interaction increases and the solubility of PEG in HFAs decreases as molecular weight (MW) increases, at least within 300-1000 Da range.73 Although the solubility of PEG with higher molecular weight has not been reported, it is reasonable to expect a similar trend in higher MW range (e.g. 1000-5000 Da). Therefore, we used ethanol (EtOH) as a cosolvent to improve mPEG5K dispersion in HFA227 propellant. EtOH is commonly used in pMDIs as an excipient to improve the solubility of drugs and excipients in HFA propellants, but usually at much higher concentrations (up to 15% v/v).36 However, a large amount of EtOH diminishes the aerosol performance of pMDI formulations,73, 74 so we sought to keep a minimum concentration of EtOH in our formulations, in this case of 0.4% v/v relative to HFA227. It is seen that the addition of small amounts of EtOH is sufficient to enhance the dispersiblity of the mPEG5K-DOX nanoparticle. In order to directly access the aerosol properties of the nanoparticle-based pMDI formulations we employ the Andersen Cascade Impactor (ACI), which is widely accepted as an in vitro lung model to determine drug deposition in the lungs. In the ACI, Stages 3 and higher represent the lower respiratory tract and deep lung region from anatomical perspective. The


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aerosol characteristics of the pMDI formulations containing mPEG-DOX nanoparticles are summarized in Figure 6 and Table 2 – raw data used to prepare that figure and to determine the aerosol parameters discussed here is show in Supporting Information - Table S1.

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It can be observed that the total fraction (and mass) of DOX deposited on Stage 3 and higher increases significantly as mPEG molecular weight decreases (mass of DOX shown in Table S1). At the same time, the amount of DOX retained in the actuator is small for the mPEG1K-DOX nanoparticle formulation, and little difference is seen in terms of the amount of DOX retained in the actuator in the nanoparticle formulations with different PEG MW. Thus, the formulation containing mPEG1K-DOX has the best aerosol quality, with respirable (RF) and fine particle fraction (FPF) of the order of 65% and 64%, respectively. We hypothesize that such high deposition efficiency is related to the enhanced dispersiblity of the particles due to high PEG density on the surface, and due to the formation of loose aggregates of nanoparticles upon evaporation of the propellant from the aerosol droplet, thus leading to the formation of micronsized aerosols, which lead to improved RF/FPF. Compared to low molecular weight mPEG, the solubility of mPEG5K in HFA decreases. To form a stable MDI formulation, a fraction of cosolvent (i.e. ethanol in this case) is added to improve mPEG5K solubility. The addition of cosolvents diminishes the respiratory fraction and fine particle fraction.73 Additionally, the mPEG5K nanoparticles tend to aggregate to microparticles (due to a lower solubility), which also results in the deposition of particles on top stages of Andersen Cascade Impactor.


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Therefore, the FPF of mPEG5K-DOX formulation was decreased. In order to put the aerosol quality reported here, typical RF and FPF reported in commercial products (drug crystals) fall within the range of RF

Nanoparticles of pH-responsive, PEG-Doxorubicin Conjugates

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