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A nano-in-nano polymer-dendrimer nanoparticlebased nanosystem for controlled multi-drug delivery Zongmin Zhao, Song Lou, Yun Hu, Jie Zhu, and Chenming Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00219 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 16, 2017

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Molecular Pharmaceutics

A nano-in-nano polymer-dendrimer nanoparticle-based nanosystem for controlled multi-drug delivery Zongmin Zhao1, Song Lou1, Yun Hu1, Jie Zhu2, Chenming Zhang1, * 1

Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, United States 2

Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, Unite States

* Correspondence to: Chenming (Mike) Zhang. Address: 210 Seitz Hall, Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA Voice: +1-(540)231-7601 Fax: +1-(540)231-3199 Email: [email protected]

May 31st, 2017

Revision to be submitted to Molecular Pharmaceutics (an ACS publication)

ACS Paragon Plus Environment


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Abstract Co-delivery of multiple chemotherapeutics with different action mechanisms is a promising strategy for cancer treatment. In this study, we developed a novel polymer-dendrimer hybrid nanoparticle-based nanosystem for efficient and controlled co-delivery of two model chemotherapeutics, doxorubicin (DOX) and paclitaxel (PTX). The nanosystem was characterized to have a nano-in-nano structure with a size of around 150 nm. The model drugs could feasibly be loaded into the nanosystem ratiometrically with high drug loading contents by controlling the feeding drug ratios. Also, the model drugs could be released from the nanosystem following a sequential release manner—specifically, quick PTX release and sustained DOX release. Acidic pH was found to enhance the release of both drugs. Moreover, the nanosystem was taken up by cancer cells rapidly and efficiently, and the delivered drugs could release sustainably and efficiently in cells to reach their action targets. In vitro cytotoxicity results demonstrated that, by optimizing drug ratios, the dual-drug-loaded nanosystem could result in better antitumor efficacy than the single-drug-loaded nanosystem or free dual-drug combination. Furthermore, the dualdrug-loaded nanosystem could induce significant changes in both the nucleus and tubulin patterns synergistically. All data suggest that the nano-in-nano polymer-dendrimer hybrid nanoparticle-based nanosystem is a promising candidate to achieve controlled multi-drug delivery for effective combination cancer therapy.

Key words: Nano-in-nano; Dendrimer; PLGA; Co-delivery; Controlled release; Synergistic effects


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1. Introduction Current chemotherapy strategies are far from ideal for cancer treatment, as they are usually associated with severe side effects, low bioavailability, and multidrug resistance.1 In recent decades, nanoparticle (NP)-based drug delivery systems have been extensively studied as a promising strategy to overcome the shortcomings of chemotherapy.2 Having many distinguishing advantages, including tunable size, modifiable surface, high loading capacity, improved drug bioavailability, controlled drug release, and precise tumor targeting, NP drug delivery systems are highly effective in maximizing therapeutic efficacy and minimizing toxicity.3-6 Many NP platforms have been designed—such as micelles, liposomes, polymeric nanoparticles, and dendrimers—and studies demonstrated that the use of these nanocarriers could significantly improve the efficacy of various chemotherapeutics.7-11

Currently, NP drug delivery systems are commonly designed for delivery of single-agent chemotherapeutics. However, the use of a single agent may suffer from some shortfalls, such as unacceptable toxicity at high drug dose and development of drug resistance, which largely limits the cancer therapeutic efficacy.12, 13 As an alternative, multi-agent therapy or combination therapy, in which two or more therapeutic agents with different action mechanisms are co-administered, has shown to be an attractive strategy over single-agent therapy.14-16 Generally, through proper drug combination, the combination therapy can promote synergistic inhibition of tumor growth by modulating different signaling pathways, improve target selectivity, maximize therapeutic efficacy, and deter the development of multidrug resistance.17, 18 As an example, it was recently reported that co-delivery of doxorubicin and dasatinib using a PEG-Fmoc nanocarrier could result in significant synergistic tumor growth inhibition both in vitro and in vivo in a breast cancer model.19 In fact, multiple combinations of agents have been investigated for cancer treatment, including chemotherapeutic drugs, phototherapeutic agents, and gene medicines.20-23 Out of these combinations, co-delivery of multiple anticancer drugs is considered a



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promising strategy to improve the therapeutic index by achieving synergistic efficacy and increasing sensitivity.24

As co-administration of free chemotherapeutic drug combinations cannot achieve the desired antitumor efficacy, NP delivery systems may provide a practical strategy for efficient delivery of multiple drugs that have dramatically different physicochemical properties and pharmacokinetic profiles. To date, some nanocarriers have been reported to successfully co-deliver different anticancer drugs.25 However, some issues still need to be solved to enhance the efficacy of co-delivery systems. First, in order to efficiently deliver drugs and minimize the side effects induced by the delivery vehicle itself, drug loading efficiency is required to be high enough.26 Also, considering the synergistic effect largely depends on the ratios of drug combinations,27 co-delivery systems should be easily engineered to enable ratiometric loading of different drugs. Moreover, as the release kinetics of different drugs may enormously affect the therapeutic outcomes of drug combinations,28 controlled release of drugs should be achieved by codelivery systems.

In this study, a novel nano-in-nano polymer-dendrimer (PD) hybrid nanoparticle-based nanosystem was designed for efficient co-delivery and controlled release of multiple chemotherapeutic drugs. As shown in the Scheme, the nanosystem was composed of smaller poly(amidoamine) (PAMAM) dendrimer NPs embedded in larger poly(lactic-co-glycolic acid) (PLGA) NP. Two model chemotherapeutic agents, doxorubicin (DOX) and paclitaxel (PTX), were loaded in the inner PAMAM NPs and the outer PLGA NP, respectively, to achieve controlled spatiotemporal release. DOX is an anthracycline agent which binds to DNAs and inhibits nucleic acid synthesis.29 PTX is a hydrophobic drug that promotes tubulin aggregation and microtubule assembly, and thus causes cell apoptosis.30, 31 Both drugs are commonly used in firstline combination cancer therapy. In this work, the nano-in-nano nanosystem was synthesized and characterized in terms of physicochemical and structural properties. In addition, the ratiometric loading


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and controlled release of dual drugs in the nanosystem were also investigated. Moreover, the uptake and intracellular distribution of the nano-in-nano nanoparticles (NPs) was studied in cancer cells, and the antitumor efficacy of the dual-drug-loaded nanosystem was evaluated in vitro in different cancer cells.

Scheme. Schematic illustration of the structure of the nano-in-nano polymer-dendrimer nanoparticlebased drug delivery nanosystem, the cellular uptake of NPs, and the controlled drug release from the nanosystem.

2. Materials and methods 2.1 Materials



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Lactel® 50:50 PLGA was purchased from Durect Corporation (Cupertino, CA). PAMAM dendrimer (generation 4), fluorescein isothiocyanate (FITC), Coumarin-6 (CM-6), Nile red, dichloromethane (DCM), and polyvinyl alcohol (PVA) were purchased from Sigma-Aldrich, Inc. (Saint Louis, MO). Ready-to-use dialysis tubes (MWCO 6000-8000) were purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA). DOX and PTX were purchased from LC Laboratories, Inc. (Woburn, MA). DAPI, Hoechst 33342, Lysotracker Red, and Tubulin Tracker Green were purchased from Life Technologies Corporation (Grand Island, NY). Fetal bovine serum, Trypsin/EDTA, F-12K medium, L-15 medium, and cancer cell lines (A-549 and MDA-MB-468) were purchased from ATCC (Manassas, VA). All other chemicals were of analytical grade.

2.2 Preparation and characterization of DOX-loaded DOX-PAMAM complex DOX was incorporated into PAMAM NPs via a film dispersion method.32 Doxorubicin hydrochloride with various molar equivalents to PAMAM was dissolved in 1.5 mL of organic solvent (chloroform: methanol=1:1). Triethylamine (TEA) (TEA: DOX= 3:1) was subsequently added to generate hydrophobic DOX. The drug solution was mixed with 10 mg of PAMAM dendrimer in 2.0 mL of organic solvent and incubated overnight at room temperature. The organic solvent was removed by vacuum evaporation to form a dry film, followed by hydrating the film with 2 mL of 0.01 M pH 7.4 PBS for 4 h. Non-incorporated DOX was removed by centrifugation at 10,000 g for 10 min (Beckman Coulter Avanti J-251, Brea, CA). The loading content of DOX in DOX-PAMAM complex was determined by measuring the fluorescence of DOX with excitation at 530 nm and emission at 590 nm according to a standard curve. For DOX-PAMAM characterization, the UV-vis spectra of free DOX, free PAMAM dendrimer, and DOX-PAMAM complex were recorded using a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT). The DOX-PAMAM complex was lyophilized and stored at 4 °C for later use.

2.3 Assembly of non-drug-loaded PD NPs


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Non-drug-loaded PD NPs were fabricated with a double-emulsion-solvent-evaporation method.33, 34 In brief, 100 mg of PLGA was dissolved in 2 mL of DCM to form the oil phase. Four hundred μL of PAMAM aqueous solution was mixed with the oil phase, and emulsified by sonication at 20% amplitude for 20 s using a sonication dismembrator (Model 500; Fisher Scientific, Pittsburg, PA). The resultant primary emulsion was added dropwise to 10 mL of 0.5 % (m/v) PVA, and was emulsified again via sonication at 60% amplitude for 120 s. The resultant secondary emulsion was stirred overnight to allow complete DCM evaporation. PD NPs were collected by centrifugation at 20,000 g for 30 min. NPs were washed three times using ultrapure water, lyophilized, and stored at 4 °C for later use.

2.4 Verification of the nano-in-nano structure of PD NPs

The nano-in-nano structure of PD NPs was characterized by positive staining transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), and Fourier transform infrared (FT-IR) spectroscopy.

For positive staining TEM imaging, NPs were dissolved in 0.01 M pH 7.4 PBS (5mg/mL) then suspended in melted agar and spun down to make pellets. The pellets were embedded in Beem® flat embedding molds (Ted Pella Inc., Redding, CA) using freshly prepared 100% Poly/Bed 812. The molds were placed in a 60 °C oven for 48 hours to cure. The embedded block was mounted in microtome specimenholder. The specimen-holder was locked into place on the trimming stage. The block surface was trimmed by a razor blade into a trapezoid or square shape as smooth and flat as possible. The trimmed samples were placed on a specimen holder which was mounted in the arm of the microtome. A diamond knife whose trough was filled with water was placed on the knife holder and adjusted to be parallel to the full face of the sample block. The samples were cut by the diamond knife to obtain sample sections. The section samples were placed on a grid, followed by processing using 2% Uranyl acetate for 10 min.



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The grid was rinsed using water and processed by lead citrate for 3 min. After rinsing the grid using water, NPs were imaged on a JEOL JEM 1400 Transmission Electron Microscope (JEOL Ltd., Tokyo, Japan).

For CLSM imaging, fluorescent PD NPs—in which PAMAM NPs were covalently labeled by FITC and PLGA layer were labeled by hydrophobic Nile red—were assembled according to a similar method as described above, except that appropriate amounts of Nile red were added into the oil phase, and FITC-PAMAM was used instead of PAMAM. The fluorescent PD NPs were suspended in 0.01 M pH 7.4 PBS (5mg/ml), and imaged on a Zeiss LSM 510 Laser Scanning Microscope (Carl Zeiss, German).

For FT-IR analysis, the spectra of PAMAM NPs, PLGA NPs, and PD NPs were recorded on a Thermo Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific Inc., Waltham, MA).

2.5 Fabrication of dual-drug-loaded PTX-DOX-PD NPs

Dual-drug-loaded PTX-DOX-PD NPs were fabricated using a similar method as PD NPs with minor modifications. In particular, 400 μL of DOX-PAMAM solution was used as the first aqueous phase and 2 mL of DCM dissolving PLGA and PTX was used as the oil phase. Dual-drug-loaded PTX/DOX-PLGA NPs, CM-6- and DOX- loaded CM-DOX-PD NPs, CM-6-loaded CM-PD NPs, PTX-loaded PTX-PD NPs, and DOXloaded DOX-PD NPs were fabricated using similar methods. NPs were washed three times with ultrapure water, freeze-dried, and stored at 4 °C for later use. To compare the loading efficiency of drugs, the same feeding contents of DOX (free DOX or DOX in DOX-PAMAM complex) and PTX were used to prepare PTX/DOX-PLGA and PTX-DOX-PD NPs, and the loading contents of drugs were determined. To prepare PTX-DOX-PD NPs with different DOX/PTX ratios, DOX-PAMAM and PTX with different feeding DOX/PTX mass ratios (5:1, 3:1, 1:1, and 1:3) were used for drug loading, and the encapsulation efficiency and loading contents of drugs were determined.


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The loading contents of drugs were determined as below. Ten mg of lyophilized NPs was dissolved in 2 mL of 0.1 N NaOH solution and incubated overnight to disrupt PLGA and thus to release the loaded drugs completely. The solution was filtered twice using a 0.22 μm filter, and the fluorescence intensity of DOX in filtrates was measured with excitation at 530 nm and emission at 590 nm. The concentration of DOX was calculated according to a standard curve. The concentration of PTX was analyzed on an HPLC system equipped with a Luna C18 (2) reverse phased chromatography column and a UV detector (at 227 nm). The encapsulation efficiency (%) was defined as (mass of loaded drug/mass of total feeding drug) *100%, and the loading content (μg/mg) was defined as (mass of loaded drug/mass of NPs).

2.6 Characterization of the physicochemical properties of NPs

Size distribution and zeta potential of NPs were analyzed on a Malvern Nano-ZS zetasizer (Malvern Instruments Ltd, Worcestershire, United Kingdom). Samples were freshly prepared before use by dispersing NPs in ultrapure water. The stability of NPs was evaluated by measuring the size change of NPs under continuous stirring at room temperature in 0.05 M citric acid buffer (pH 5.0), 0.01 M PBS (pH 7.4), and 10% (v/v) human serum.

2.7 Testing the release kinetics of drugs in vitro

In vitro release kinetics of DOX and PTX were measured using a dialysis method. In brief, 10 mg of NPs were dissolved in buffers and dialyzed against 25 mL of buffers (0.01 M PBS at pH 7.4 and 0.05 M citric acid buffer at pH 5.0, with 0.1% [v/v] Tween 80 supplemented) using ready-to-use dialysis tubes (MWCO 6000-8000). The release testing was conducted under continuous stirring at 37 °C. At predetermined time points, a 1 mL sample was taken out, and equal volumes of fresh buffer were added. The concentration of DOX and PTX was determined by fluorescence-measuring and HPLC as described above, respectively.



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2.8 Analysis of the uptake and intracellular distribution of NPs in cells

MDA-MB-468 breast cancer cells and A549 lung cancer cells were cultured in L-15 medium and F-12K medium, respectively, in a humidified atmosphere at 37 °C. The culture media for both cells were supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% fetal bovine serum.

The uptake of NPs was quantitatively measured by flow cytometry in MDA-MB-468 cells. CM-6 labeled CM-PD NPs were fabricated by adding CM-6 in the oil phase during the preparation process. Cells were seeded into 24-well plates (CORNING, Tewksbury, MA) at a concentration of 2×106/well, and cultured for 24 h. The original medium was replaced with fresh medium containing 20 μg of CM-PD NPs. After incubation for 1 h, 2 h, 4 h, 6 h, 8 h, or 12 h, the medium was immediately removed, and cells were washed three times using 0.01 M pH 7.4 PBS. Cells were then detached from culture plates using Trypsin/EDTA solution and centrifuged at 200 g for 10 min, and cell pellets were re-suspended in 0.01 M pH 7.4 PBS. Samples were immediately analyzed on a flow cytometer (BD FACSAria I, BD, Franklin Lakes, NJ). A similar method was used to quantitatively compare the uptake of PTX/DOX-PLGA and PTX-DOX-PD NPs, except that cells were treated with the NPs based on containing same amounts of DOX (0.025 μM) for 4 h or 12 h.

The uptake and intracellular distribution of NPs were studied by CLSM towards A549 or MDA-MB-468 cells. Cells were seeded onto a 2-well chamber slide (Thermo Fisher Scientific Inc., Rockford, IL, USA) at a concentration of 2×105/chamber in 2 mL of medium, and cultured overnight. The original medium was then replaced with 2 mL of fresh medium. To study the cellular uptake of NPs, cells were incubated with 20 μg of CM-DOX-PD NPs for 1 h, 2 h, 4 h, 6 h, or 12 h. To compare the uptake of free CM-6+DOX, CM/DOX-PLGA NPs, and CM-DOX-PD NPs, cells were treated with different drug formulations containing the same amounts of CM and DOX for 4 h. The medium was removed and cells were washed three times with PBS. One mL of freshly-prepared 4% (w/v) paraformaldehyde was added to each well to fix the cells


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for 15 min. The fixed cells were permeabilized by adding 0.5 mL of 0.1% (v/v) Triton™ X-100 for 15 min. After washing the cells three times again, the nuclei were stained with DAPI according to the standard protocol provided by the supplier. To study the intracellular distribution of NPs, cells were treated with 50 μg of CM-6 labeled CM-PD NPs for 4 h and 8 h. The lysosome and nucleus of cells were labelled by Lysotracker Red and Hoechst 33342, respectively. The intracellular distribution of NPs was visualized on a Zeiss LSM 510 Laser Scanning Microscope. To observe the tubulin pattern in vitro, cells were incubated with drug formulations containing 0.5 μM of total drugs for 12 h. Tubulin Tracker Green (200 nM) were mixed with cells for 1 h at 37 °C to label tubulin, and subsequently cell nuclei were labeled by Hoechst 33342 for 10 min. Cells were washed three times and immediately observed in the buffer.

2.9 In vitro cytotoxicity assay

In vitro cytotoxicity of drug formulations was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Cells were seeded into 96-well plates at a density of 5000/well and incubated for 24 h. The original medium was replaced with fresh medium and cells were treated with different concentrations of drug formulations. After incubation for 72 h, the medium was replaced with fresh medium containing MTT (0.5 mg/mL), and cells were incubated for another 4 h at 37°C. The medium was removed immediately, followed by adding 100 μL of DMSO to solubilize the formed formazan crystal. Absorbance was measured at 570 nm using a Synergy HT Multi-Mode Microplate Reader. Cell viability was calculated using untreated cells as a control. The inhibitory concentration (ICx), which is the drug concentration to produce x% cell death, was calculated using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA). The combination index (CIx) was calculated using the Chou and Talalay’s method35 according to the following equation:

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(‫)ܦ‬ଵ (‫)ܦ‬ଶ + (‫ܥܫ‬௫ )ଵ (‫ܥܫ‬௫ )ଶ



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(ICx)1 and (ICx)2 are the ICx of DOX-loaded formulation and PTX-loaded formulation, respectively. (D)1 and (D)2 are the concentration of DOX and PTX in the dual-drug-loaded formulation at the ICx value.

To study the morphological change of cells treated with drug formulations, MDA-MB-468 cells were seeded into a 12-well plate and cultured for 24 h. Cells were incubated with drug formulations containing 5 μM of total drugs for 24 h. Cells were then washed and their morphology was observed using a Nikon Eclipse E600 light microscope and images were captured using a Nikon DS-Fi1 camera.

2.10 Statistical analysis

All experiments were performed at least in triplicate. Data were expressed as means ± standard deviation. Significance tests between two groups or among multiple groups were conducted using student’s t-test or one-way ANOVA followed by Tukey’s HSD analysis, respectively. Differences were considered to be significant at P-values < 0.05.

3. Results and discussion 3.1 Characterization of the nano-in-nano structure of polymer-dendrimer hybrid NPs

In this study, the proposed polymer-dendrimer NP-based nanosystem is conceptualized to have a nanoin-nano structure in which different drugs can be carried by different compartments to achieve controlled spatiotemporal release. Here, CLSM was used to verify the successful formation of the nanoin-nano structure. PAMAM NPs were covalently labelled by FITC, and PLGA NPs were labeled by Nile red. As shown in Fig. 1A, the majority of particles were co-labelled by FITC and Nile red, indicating that PAMAM NPs were successfully loaded to form the nano-in-nano structure. Meanwhile, as shown in Fig. S1, single dye labeled PD nanoparticles did not show the fluorescence of the other dye, excluding the possibility that the co-localization of dual colors was caused by fluorescence excitation interactions. The FT-IR results shown in Fig. 1B further confirmed the successful embedding of PAMAM NPs in PLGA NPs.


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Specifically, a peak at 1752 cm-1, which is contributed by the C=O stretching of carboxylic groups of PLGA, was observed in the spectra of both PLGA NPs and PD hybrid NPs. In addition, a peak at 1557 cm-1 which is corresponding to the C=O stretching (amide I) of PAMAM showed in the spectra of both PAMAM and PD hybrid NPs.

The morphology of NPs was visualized by TEM. As shown in Fig. 2A, all three NPs, including PLGA NPs, non-drug-loaded PD NPs, and dual-drug-loaded PTX-DOX-PD NPs, were of spherical shapes. Meanwhile, black dots which may be formed by PAMAM NPs were observed in PD and PTX-DOX-PD NPs but not in PLGA NPs, further suggesting that multiple PAMAM dendrimer particles were successfully encapsulated into one hybrid NP. As shown in Fig. 2B, the average size of the three NPs was measured to be 125.7 ± 5.2 nm, 141.9 ± 3.4 nm, and 164.2 ± 4.7 nm, respectively, suggesting that the loading of PAMAM NPs or drugs would slightly increase the size of NPs. Moreover, Fig. 2C shows that PD and PTX-DOX-PD NPs exhibited a narrow size distribution, which was consistent with the low PDI indexes, which were measured to be 0.24 ± 0.07 and 0.19 ± 0.04, respectively (Fig. 2B). The zeta potential of PLGA, PD, and PTX-DOX-PD NPs was -10.69 ± 0.45 mV, -7.02 ± 0.27 mV, and -5.42 ± 0.76 mV, respectively (Fig. 2B). As it has been reported that particles smaller than 200 nm with hydrophilic surfaces tend to show enhanced accumulation in tumor sites mediated by the EPR effect,9 the polymer-dendrimer NPs thus have relatively optimal physicochemical properties for efficient delivery of multi-drugs to tumor sites.



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Fig. 1. CLSM and FT-IR characterization of the polymer-dendrimer hybrid NPs. (A) CLSM images of fluorescently labeled PD NPs, in which PAMAM dendrimer NPs were covalently labeled by FITC and PLGA layer was labeled by Nile red. The left and right panels show the zoom-out and zoom-in images of NPs, respectively. (B) FT-IR spectra of PLGA NPs, PAMAM NPs, and PD hybrid NPs.


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Fig. 2. Characterization of the morphological and physicochemical properties of NPs. (A) TEM images showing the structure of (a) PLGA NPs, (b) PAMAM NPs, (c) non-drug-loaded PD NPs, and (d) dual-drugloaded PTX-DOX-PD NPs. All scale bars represent 100 nm. (B) and (C) show the physicochemical properties and size distribution of PLGA, PD, and PTX-DOX-PD NPs, respectively.

3.2 Characterization of DOX-PAMAM complex

As shown in Fig. 3A, UV-vis results demonstrated that a specific absorbance peak at 491 nm was observed in the spectrum of DOX-PAMAM complex, demonstrating the successful loading of DOX into PAMAM NPs. The influence of feeding DOX/PAMAM ratios on the DOX loading was further studied (Fig.



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3B). Up to 315.7 μg/mg DOX can be loaded into the DOX-PAMAM complex with high encapsulation efficiency (79.1%) when using a feeding DOX/PAMAM ratio of 15:1. Noticeably, with more DOX loaded, both the size and zeta potential of DOX-PAMAM complex increased accordingly.

3.3 Fabrication of dual-drug-loaded PTX-DOX-PD NPs with precise ratiometric drug loading

The impact of PAMAM on the drug-loading contents in NPs were studied. Two independent tests (denoted as Test A and Test B) were conducted. In each test, PTX/DOX-PLGA and PTX-DOX-PD NPs were prepared using the same feeding contents of DOX and PTX. Test A and B had a same overall drug feeding content but different PTX/DOX feeding ratios. As shown in Fig. 3D, in both tests, the PTX loading contents in PLGA and PD NPs were similar. However, the DOX loading contents were significantly higher in PD NPs than in PLGA NPs, suggesting that the design of nano-in-nano nanosystem (PAMAM in PLGA) could enhance the loading of DOX due to the high drug loading capacity of PAMAM (Fig. 3B). In order to study the feasibility of preparing ratiometric-drug-loaded PTX-DOX-PD NPs, multiple tests were carried out in which DOX-PAMAM and PTX with different DOX/PTX feeding ratios (5:1, 3:1, 1:1, and 1:3) were used for dual-drug-loaded nanoparticle preparation. As shown in Fig. 3C, various loading contents of DOX and PTX could be encapsulated in the nanosystem with very high efficiency (over 70%). The real ratios of loaded DOX/PTX were measured to be 4.64:1, 2.64:1, 0.84:1, and 1:3.11, indicating that the loaded drug ratios in the nanosystem could readily be adjusted by controlling the feeding drug ratios. The relative magnitude of different signaling pathways in cancer cells can be affected by the relative ratios of administered drugs, and drug synergistic effects can be induced only at certain relative magnitudes of signaling pathways.36-38 Therefore, an appropriate drug ratio in a co-delivery nanosystem is a necessity in achieving drug synergy in cancer cells.27, 39 In this study, the nanosystem was loaded with drugs of different ratios, providing the possibility for screening drug ratios to achieve a potent synergistic effect.


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The stability of dual-drug-loaded PTX-DOX-PD NPs was studied in three different buffers. As shown in Fig. 3E, PTX-DOX-PD NPs were most stable in 0.01 M pH 7.4 PBS, having the slowest size increase over time. In the other two buffers (0.05 M pH 5.0 citric acid buffer and 10% human serum), the size of NPs changed dramatically over time, showing to be less stable than in pH 7.4 PBS.



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Fig. 3. Characterization of DOX-PAMAM complex and dual-drug-loaded PTX-DOX-PD NPs. (A) UV-vis spectra of PAMAM, DOX, and DOX-PAMAM complex. (B) The influence of feeding DOX/PAMAM ratio on the encapsulation efficiency and loading content of DOX in DOX-PAMAM complex. (C) Characterization of different drug formulations. In (B) and (C), E.E represents encapsulation efficiency, and L.C represents loading content. (D) Comparison of the loading contents of drugs in PTX-DOX-PD and PTX/DOX-PLGA NPs. * p < 0.05, ** p < 0.01 (student’s t-test). (E) Stability of PTX-DOX-PD NPs in different buffers.

3.4 In vitro release kinetics of drugs from NPs

The in vitro release kinetics of DOX and PTX from different NPs were tested at both physiological pH (pH 7.4) and lysosomal pH (pH 5.0) at 37 °C. As shown in Fig. 4, the release of both PTX and DOX from dualdrug-loaded PLGA NPs (PTX/DOX-PLGA) followed a similarly fast release manner at both pH 7.4 and pH 5.0; pH values did not significantly affect the release of drugs. In contrast, the releases of PTX and DOX from dual-drug-loaded PD NPs (PTX-DOX-PD) were different. PTX released quickly, in a similar manner as that from PTX/DOX-PLGA NPs. However, the release of DOX from PTX-DOX-PD NPs followed a more controlled and sustained release pattern compared to that from PTX/DOX-PLGA NPs and DOX-PAMAM complex at both pH 7.4 and pH 5.0 (Fig. 4A and Fig. 4B). Interestingly, the DOX release from DOXPAMAM NPs, and the DOX and PTX release from PTX-DOX-PD NPs, were more rapid and efficient at pH 5.0 than at pH 7.4. This evidence may be explained by the proton sponge effect of PAMAM.32, 40 At lower pH values, the tertiary amine groups (pKa=6.4) of PAMAM were protonated,41 generating intense electrostatic repulsion among dendrimer branches, thus leading to enhanced DOX release. Also, the repulsion between individual protonated dendrimer NPs might cause PTX-DOX-PD NPs to be less stable (Fig. 3E), thus promoting the release of PTX. This pH-dependent release of DOX and PTX may reduce premature basal drug release, reduce undesired side toxicity, and increase the bioavailability to tumor sites.


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The comparison of the release kinetics of dual drugs from PTX/DOX-PLGA NPs and PTX-DOX-PD NPs suggested that the controlled sequential release of dual drugs—specifically, quick release of PTX followed by sustained release of DOX—can be attributed to the fact that drugs were located in different compartments of the unique nano-in-nano structure. In PTX/DOX-PLGA NPs, as both DOX and PTX were loaded in PLGA NPs, the two drugs exhibited a simultaneous fast release profile. However, in PTX-DOXPD NPs, PTX was loaded in the out PLGA nanoparticle compartment and only had one barrier to diffuse through (PLGA). DOX was located in the inner PAMAM nanoparticle compartment and had to diffuse through two barriers (PAMAM and PLGA). Therefore, PTX and DOX released in a controlled sequential release manner instead of a simultaneous quick release one. The earlier fast released PTX was expected to sensitize the tumor microenvironment, such as resulting in tumor cell apoptosis, increasing interstitial space, and decreasing the interstitial fluid pressure,28,

42, 43

thus facilitating the penetration of

nanoparticles deeply into tumors and enhancing the bioavailability of drugs. On the other hand, DOX would continuously be released and interact with DNA, resulting in improved antitumor efficacy.



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Fig. 4. In vitro release kinetics of loaded drugs. (A) Release profile of DOX at pH 5.0. (B) Release profile of DOX at pH 7.4. (C) Release profile of PTX at pH 5.0. (D) Release profile of PTX at pH 7.4. (E) Release profiles of PTX and DOX from dual-drug-loaded PTX-DOX-PD NPs showing in an integrated graph.

3.5 Cellular uptake of nano-in-nano polymer-dendrimer NPs

Efficient uptake of drug-loaded NPs by cancer cells is a necessity for the success of drug delivery. Herein, the uptake of CM-PD NPs, in which CM-6 that is a hydrophobic fluorescent dye was used to label NPs, was quantitatively examined by flow cytometry, and the results are shown in Fig. 5A and Fig. 5B. After


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incubating cells with NPs for 2 hours, 99.8% of the studied cells were stained by CM-6 fluorescence (data not shown), suggesting that cells could rapidly take up polymer-dendrimer NPs in a short period. In addition, the NP uptake kinetics (Fig. 5B) revealed that cells could continuously take up the nano-in-nano NPs in the first 8 hours, and the process was saturated after that. The rapid and efficient uptake of NPs may increase the bioavailability and enhance the cytotoxicity efficacy of delivered drugs.

The cellular uptake of polymer-dendrimer NPs by cancer cells was also visualized using CLSM. Cells were treated with CM-DOX-PD NPs for up to 12 hours. According to Fig. 5C, the uptake of NPs was timedependent, which is consistent with the flow cytometry results shown in Fig. 5A and Fig. 5B. With the incubation time increasing, more NPs were observed to accumulate in cells. Noticeably, at 1 and 2 h, the red and green fluorescence were almost co-localized in the cytosol (shown as yellow) and no red DOX fluorescence was observed in the nucleus area, indicating no substantial DOX had been released from NPs. In contrast, from 4 h, more DOX fluorescence was accumulated in the nucleus, suggesting that DOX was continuously released from NPs to reach its action targets. Moreover, at 12 h, almost all DOX was found to be in the nucleus area, revealing that DOX had been efficiently released from NPs. Noticeably, this complete DOX release at 12 h in cells seemed to be different from the in-buffer release data, as only around 12% of DOX had released at 12 h in the buffer of pH 5.0. This discrepancy may be attributed to the fact that there are many hydrolases and other enzymes inside cells, especially in the lysosomes. These enzymes would destruct and degrade drug-loaded NPs to lead to a faster release of drugs.

The intracellular release and distribution of drugs were also compared by incubating cells with different drug formulations. Cells were treated with CM-DOX-PD NPs, CM/DOX-PLGA NPs, or free CM+DOX for 4 hours, and the state of drug distribution in cells was observed using CLSM. As shown in Fig. 6A, compared to the treatment with free CM+DOX, more green fluorescence was internalized in cells treated with CM/DOX-PLGA NPs and CM-DOX-PD NPs, indicating that the use of NP as a vehicle would enhance



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the uptake efficacy of hydrophobic drugs. Interestingly, it was found that most DOX had released from CM/DOX-PLGA NPs and accumulated in the nucleus after 4 hours. However, DOX showed to be in both cytosol and nucleus after treated with CM-DOX-PD NPs within the same time frame, suggesting that the intracellular release of DOX from polymer-dendrimer NPs was more sustained than that from PLGA NPs, which is in agreement with the in vitro drug release results shown in Fig. 4. The sustainably released DOX may constantly interact with DNA intracellularly for an extended period, and may enhance the cytotoxicity of delivered drugs. As PTX-DOX-PD NPs had a higher drug loading content over PTX/DOXPLGA NPs (Fig. 3C), the drug delivery efficiency of those two NPs was compared using flow cytometry. Cells were treated with PTX-DOX-PD NPs or PTX/DOX-PLGA NPs based on containing same amounts of DOX (0.025 μM), and the intracellular DOX fluorescence intensity was studied. As shown in Fig. 6B and Fig. 6C, the mean fluorescence intensity (M.F.I) of DOX in PTX-DOX-PD group was significantly higher than that in the PTX/DOX-PLGA group at both 4 h and 12 h, suggesting that the high drug loading content would enhance the intracellular drug delivery efficiency. The improved drug delivery efficiency can increase the accumulation of drugs in cells, thus leading to enhanced cytotoxicity efficacy. In addition, as shown in Fig. S2, the DOX/CM fluorescence intensity ratios in cells after being treated with CM-DOX-PD NPs for 4 or 8 hours were similar to that of CM-DOX-PD NPs. This suggested that the dual model drugs would be delivered into cells maintaining a similar ratio as in the dual-drug-loaded nanosystem. Future work needs to examine the drug ratios delivered to tumor tissues in vivo.

The intracellular distribution of NPs after internalization was also studied. Cells were incubated with CMPD NPs for 2 or 8 h, and their endosomes/lysosomes were labeled by Lysotracker Red. As shown in Fig. 7, at 2 h, most green fluorescence was co-localized with the red fluorescence, revealing that the internalized NPs were possibly translocated through lysosome pathways after entering cells. As there are many hydrolases and other enzymes in lysosomes,16 NPs can be destructed and degraded rapidly to induce the release of loaded drugs. Meanwhile, the acidic pH in lysosomes could also facilitate drug


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release from PTX-DOX-PD NPs. At 8 h, substantial amounts of green fluorescence did not overlap with the red fluorescence, indicating that substantial amounts of CM-6 escaped from lysosomes. After escaping from lysosomes, drugs may be promoted to reach their target functioning sites to start their actions.



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Fig. 5. Cellular uptake of polymer-dendrimer hybrid NPs in cancer cells. (A) The intensity distribution of CM-6 in MDA-MB-468 cells after treated with 20 μg of CM-PD NPs for up to 12 hours. (B) M.F.I of CM-6 corresponding to (A). (C) CLSM images showing the cellular uptake of CM-DOX-PD NPs at different time points. A549 cells were treated with 20 μg of CM-6- and DOX- loaded CM-DOX-PD NPs for 1 h, 2 h, 4 h, 6 h, or 12 h. Scale bars represent 10 μm.

Fig. 6. Comparison of the cellular uptake of dual-drug-loaded PLGA and PD NPs. (A) Comparison of the cellular uptake of free CM-6+DOX, CM/DOX-PLGA NPs, and CM-DOX-PD NPs in MDA-MB-468 cells after 4


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hours’ treatment. Scale bars represent 10 μm. (B) Flow cytometry analysis showing the intensity distribution of DOX in MDA-MB-468 cells treated with PTX/DOX-PLGA NPs or PTX-DOX-PD NPs based on containing same amounts of DOX (0.025 μM) for 4 h or 12 h. (C) M.F.I of DOX corresponding to (B). Significantly different: * p

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