Magnetic Fluorescent Nanoformulation for Intracellular Drug Delivery

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Magnetic Fluorescent Nanoformulation for Intracellular Drug Delivery to Human Breast Cancer, Primary Tumors, and Tumor Biopsies: Beyond Targeting Expectations Kheireddine El-Boubbou,*,†,‡,§ Rizwan Ali,‡,§ Hassan M. Bahhari,‡ Khaled O. AlSaad,‡ Atef Nehdi,‡ Mohamed Boudjelal,‡ and Abdulmohsen AlKushi† †

King Saud bin Abdulaziz University for Health Sciences (KSAU-HS), King Abdulaziz Medical City, National Guard Health Affairs, Riyadh 11481, Saudi Arabia ‡ King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, National Guard Hospital, Riyadh 11426, Saudi Arabia S Supporting Information *

ABSTRACT: We report the development of a chemotherapeutic nanoformulation made of polyvinylpyrrolidonestabilized magnetofluorescent nanoparticles (Fl-PMNPs) loaded with anticancer drugs as a promising drug carrier homing to human breast cancer cells, primary tumors, and solid tumors. First, nanoparticle uptake and cell death were evaluated in three types of human breast cells: two metastatic cancerous MCF-7 and MDA-MB-231 cells and nontumorigenic MCF-10A cells. While Fl-PMNPs were not toxic to cells even at the highest concentrations used, Dox-loaded FlPMNPs showed significant potency, effectively killing the different breast cancer cells, albeit at different affinities. Interestingly and superior to free Dox, Dox-loaded Fl-PMNPs were found to be more effective in killing the metastatic cells (2- to 3-fold enhanced cytotoxicities for MDA-MB-231 compared to MCF-7), compared to the normal noncancerous MCF-10A cells (up to 8-fold), suggesting huge potentials as selective anticancer agents. Electron and live confocal microscopy imaging mechanistically confirmed that the nanoparticles were successfully endocytosed and packaged into vesicles inside the cytoplasm, where Dox is released and then translocated to the nucleus exerting its cytotoxic action and causing apoptotic cell death. Furthermore, commendable and enhanced penetration in 3D multilayered primary tumor cells derived from primary lesions as well as in patient breast tumor biopsies was observed, killing the tumor cells inside. The designed nanocarriers described here can potentially open new opportunities for breast cancer patients, especially in theranostic imaging and hyperthermia. While many prior studies have focused on targeting ligands to specific receptors to improve efficacies, we discovered that even with passive-targeted tailored delivery system enhanced toxic responses can be attained.

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INTRODUCTION The hoped “cure” for cancer has proven far more indescribable than anticipated, making this complex set of diseases one of the leading causes of death worldwide.1 The most deadly aspect of this complex disease is its ability to spread or metastasize, eventually clustering into a primary tumor, where the cancerous cells may begin to break off and invade the whole body. Owing to the multidrug-resistance factor,2 a wide range of anticancer drugs can be expelled out of cancer cells, preventing their intracellular accumulation to cytotoxic levels. Thus, the efficiency of systematic chemotherapeutics is reduced, leading to serious off-target side effects.3 This challenge has been a major cause leading to failure of tumor chemotherapy clinically. Despite the continuous efforts to combat such metastatic cases, chemotherapeutic formulations and nanoparticulate drug delivery systems have shown limited success, without effective translation into clinic. To address these limitations, one © XXXX American Chemical Society

approach is to design a rapid delivery system, preferably magnetoreceptive, that either delivers NPs into the target cells by vesicle-mediated endocytosis/transcytosis or keeps the NPs in the microvessels of the target cell, which usually occurs within a few hours.4 This is expected to enhance therapeutic efficacy and deliver cytotoxic doses of drugs intracellularly. Furthermore, it would be highly desirable that the delivery process can be magnetically imaged and optically tracked simultaneously, offering dual-modal probes which can provide useful information regarding drug uptake and biodistribution.5,6 Recently, innovative nanotherapeutic approaches beyond the conventional therapies of using chemical drugs are being actively developed. By nanosizing a formulation, one can carry a Received: February 22, 2016 Revised: May 25, 2016

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DOI: 10.1021/acs.bioconjchem.6b00257 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Scheme 1. Schematic Illustration of the Synthesis of Dox-Loaded Fluoresceinated PMNPs

(PEG) and a variety of their copolymers.24−26 In particular, the old-rooted water-soluble PVP has been widely used as a biomaterial in medicine as well as in pharmaceutical production in tablets (i.e., Leflumax), oral solutions, and injectables.27 One of the most key features of PVP is its amphiphilicity and universal solubility in water due to its polar lactam group in the pyrrolidone ring. This allows PVP to form water-soluble complexes with many active substances, increasing their dissolution and hence enhancing their bioavailability.28,29 Thus, owing to their hemocompatibility, tissuecompatibility, and low toxicity, PVP-grafted MNPs (as alternative to PEGcoated formulations) are recently being developed for cellular uptake, imaging, and delivery applications in vitro and in vivo.30−35 Besides all that, for optimal capacity to incorporate chemotherapeutic drugs and have effective cellular internalization, studies have shown that the coated MNPs should have average hydrodynamic sizes of 10−150 nm and ζ potential values in the range of −40 to +15 mV. Small particles (150 nm) will be mainly captured by macrophages in reticuloendothelial system or cleared by Kupffer cells in the liver before they reach their targets. Nevertheless, up to now, syntheses of such high-quality stable colloidal MNPs that deliver the drugs to the target efficiently in a controlled-manner are still challenging. In this work, a chemotherapeutic nanoformulation made of fluorescently labeled PVPylated MNP (Fl-PMNP) loaded with the anticancer drug Doxorubicin (Dox) was developed, and its effect on the intracellular delivery and killing of different types of human breast cells as well as penetration to 3D primary tumors and solid tumors was explored. We focused on two breast metastatic adenocarcinoma cell lines MCF-7 and MDAMB-231 and a nontumorigenic MCF-10A breast cells having different characteristics, endocytic potentials, and response to chemotherapy. Furthermore, primary breast tumors derived from primary lesions rather than from more aggressive and metastatic tumors were also tested. We found this is essential in our study to reflect the effect of nanoparticle uptake and efficacies on the various stages of tumor progression from early stage breast cancers to late-stage progressive models. Importantly, enhanced penetration through patient tumor tissue was also observed, pinpointing the potential of an important clinical improvement of chemotherapy to tumors. The Dox-loaded Fl-PMNPs used here can potentially open new opportunities for in vivo breast cancer therapeutic imaging and hyperthermia. To our knowledge, this is the first report using PVPylated MNP formulation to systematically study the delivery of cytotoxic agents to different types and stages of human breast cancer cells and tumors.

pharmaceutical drug, increase its dissolution rate, direct its delivery, and enhance its local concentration, giving promise for next-generation cancer treatment. In addition, inexpensive but safe materials have to be explored to reduce the cost of the nanobased therapy so that a greater patient population can benefit. Of the various drug delivery systems,7 we have become interested in the development of multifunctional magnetic nanoparticles (MNPs) for targeted imaging and drug delivery to cancerous cells.8−10 Owing to their large surface area to volume ratios, ease of synthesis, surface functionalities, tunable sizes, and low costs, MNPs have shown to be highly desirable for both therapeutics and diagnosis. With their excellent magnetic abilities, high biocompatibilities, and low toxicities, MNPs can serve as both magnetic resonance imaging (MRI) probes and effective delivery vehicles. Several iron-based MNPs such as Feridex, Sinerem, or Resovist have been already approved by FDA for use in humans, mainly as MRI liver contrast agents.11 Coassembling fluorescent and therapeutic drugs onto MNPs offers the possibility of producing new classes of supernanoparticles with a set of combined potentials for in vivo photon and MR imaging. Previous reports have demonstrated the utilization of drug-loaded MNPs for cancer imaging and therapy.12 Mostly, many studies showed that there is a significant role of MNPs not only in enhancing intracellular drug uptake but also in offering possible means to inhibit/ reverse the cellular drug resistance, thus providing promising potentials in cancer theranostics.13,14 However, the hampered clinical translation necessitates identification of specific nanoformulation designs that are critical to performance, especially when required for biomedical and clinical end points. Thus, the following design criteria need to be fulfilled: ultrasmall sizes, high magnetic contrast, long-term colloidal stability, biocompatibility, bioavailability, and maximized loading of biomolecules and drugs. Because of the leaky endothelium and vascularate in cancerous tissues that are absent in normal tissues, significant and increased localization of NPs can be significantly achieved by what is known as the enhanced permeability and retention (EPR) effect.15 Coating with biocompatible polymers provides good steric hindrance and protection to the formulation and minimizes protein binding to the surface of NPs (i.e., opsonization), offering prolonged circulating NPs and better targeting efficacies.16 This is crucial, as it will provide a stable injectable colloidal solution or a dried powder that is easy to reconstitute. Many synthetic polymers have been coated on NP surfaces and are actively proposed as drug carriers including but not limited to polysaccharides (hyaluronan, dextran, etc.),17,18 polyvinyl alcohol (PVA),19 polyacrylic acid (PAA),20 polyvinylpyrrolidone (PVP),21,22 poly(lactic-co-glycolic acid) (PLGA), 2 3 poly-N-2hydroxypropylmethacrylamide (HPMA), polyethylene glycol B


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Bioconjugate Chemistry

Figure 1. Physiochemical properties of Dox@Fl-PMNPs. (A) DLS measurement of Dox@Fl-PMNP showing hydrodynamic size (DH = 80 nm). (B) ζ-Potential of the different NP constructs (bare-MNP, Fl-PMNP, and Dox@Fl-PMNP) suspended in water. (C) FTIR spectra for Dox@Fl-PMNP (above) and free Dox (below), clearly showing the peaks associated with Dox and its successful loading. (D) UV−vis spectra of the different NP aqueous dispersions, clearly showing the successful loading of Fl and Dox, respectively (inset: optical detection of Fl-PMNP and Dox-loaded FlPMNPs illuminated by a portable UV lamp).

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RESULTS AND DISCUSSION Synthesis and Characterization of Drug-Loaded Fluoresceinated PVPylated Iron Oxide Nanoparticles. The step-by-step methodology used to prepare Dox-loaded FlPMNPs is outlined in Scheme 1. Our studies demonstrated that this simple approach formed colloidal water-dispersible drugloaded nanoformulation stable for months, without any loss of physiochemical properties. We hypothesized that the PVP polymer would anchor at the interface to confer an aqueous dispersity to the formulation and the amphiphilic drug would intercalate into the PVP coat complexing the fluorescent dye. Due to its random-coil structure in aqueous solutions, PVP forms complexes with low molecular weight hydrophilic and hydrophobic compounds and drugs, especially H-donors and aromatic molecules.36,37 In particular, PVP has been shown to complex Fl, forming a 1:1 complex at low pH values (3−5) when the ratio of Fl to PVP is small.38 Moreover, adsorption of carboxylic acids to the iron/iron oxide surface in a chelating bidentate mode is well-known.39,40 Furthermore, pyrrolidone moieties have been proven to coordinate to magnetite nanocrystal surfaces rendering them water-soluble and colloidal.41 In fact, PVP acts as a stabilizer for dissolved metallic salts through steric and electrostatic stabilization of the amide groups of the pyrrolidone rings. Inspired by these observations and based on our previously published work using the in situ precipitation (Ko-precipitation) hydrolytic basic (KHB) methodology,40 we designed the Fl-PMNP formulation.

Briefly, sequential Ko-precipitation of iron salts in the presence of premixed fluorescein and PVP afforded exceptionally stable colloidal Fl-PMNPs suspended in water. As the fluoresceinated PMNP was excellently dissolved in aqueous media forming colloidal dispersions stable for months, it is most probable that the fluoresceinated PVP is strongly chelated to the iron oxide via coordinating (through carbonyl and carboxylate groups) and noncoordinating PVP fragments, as has been suggested earlier.42 This unique combined feature offers super dispersibilty and stabilization to the formed nanoconstructs. Next, simple adsorption of Dox onto the labeled Fl-PMNP was chosen, as it has the advantage to preserve both the structure of the NPs and the attached Dox. Up to 57% of the drug was loaded onto Fl-PMNP (i.e., 135 mg of Dox/g of FlPMNPs), as evident from absorption spectroscopy (Figure S1 in Supporting Information). MNPs where drug molecules are covalently conjugated to the NPs’ surface usually exhibit low drug entrapment efficiencies and more difficulty in drug release at the target site due to the covalent binding.10,43 Furthermore, the orientation of the active functional moieties present in drugs is extremely important. For instance, when carboxylic acid-functionalized NPs interact with ligands with multiple amine groups, inactivation of ligands might be observed. Thus, we envisioned that electrostatic, hydrogen bonding, and van der Waals interactions might be a better choice to preserve the activity of the drug on the NPs. C


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pH = 4.5 and 40% at pH = 7.4. This step was followed by a more controlled slower release, with ∼80% discharge at pH = 4.5 and only 55% at the physiologically neutral pH. This rapid but sustained release of Dox is unique and probably preferable to reach cytotoxic concentrations inside the cells, for both in vitro and in vivo cancer therapy, as has been seen earlier.46−49 In another fact, it is worth pinpointing that acetaminophen tablets complexed with 4% PVP as binder released the drug more quickly than tablets with gelatin or hydroxypropylcellulose (this also applies to other sustained-release PVP matrix tablets).29 All this implies that in our system the physically bound drug molecules could be released rapidly, proficiently, and sustainably, making Fl-PMNP a promising candidate as a pharmaceutical carrier for imaging, drug delivery, and therapy. Uptake and Intracellular Distribution of Dox@FlPMNPs. We first examined uptake of the NPs with the three different breast cell lines using flow cytometry. Upon incubation of the cells with Fl-PMNPs (10 μg/mL), all cell types showed significant cellular uptake as time elapses (Figure 2A). The uptake was rapid with no significant changes after 4 h.

The Dox-loaded Fl-PMNPs obtained were thoroughly characterized by a variety of techniques including DLS, FTIR, TEM, and TGA (Figure 1). The NPs were homogeneous with an average core size of 5 nm, hydrodynamic size (DH) of 80 nm, pinpointing the narrow size distribution and uniformity of the as-synthesized particles (Figure 1A). DLS measurements were also recorded after 6 months showing no significant change in the size, further confirming the remarkable stability of the particles in their aqueous dispersions. We believe that this extra stability is due to the π−π stacking of the aromatic rings of fluorescein and Dox, similar to an earlier observation.44,45 Zeta potential measurements indicated that loading with Dox onto Fl-PMNP causes a shift in ζ potential from −10 mV to +5 mV, as shown in Figure 1B and Figure S2. FTIR spectra clearly showed that the Dox@Fl-PMNPs have the same characteristic absorption bands similar to the free Dox (Figure 1C), with distinctive shifted and overlapped peaks at ∼3400 cm−1 due to O−H and N−H stretching vibrations. Peaks at 2920, 2835 cm−1 are due to PVP, Dox, and fluorescein C−H stretching vibrations. Fe−O stretching at ∼560 and 610 cm−1 confirmed the presence of iron oxide in the core of the construct. Importantly, the spectrum showed the disappearance of the band at 1731 cm−1 corresponding to C-13 carbonyl of Dox. Moreover, the N−H stretching vibrations of the primary amine at ∼3520 cm−1 and the bands observed at ∼805 cm−1 due to the N−H wag in pure Dox seem to disappear in the spectrum of Dox@Fl-PMNPs. All this indicates that the attachment of Dox to Fl-PMNP occurs via the interaction of −NH2, −OH, and carbonyl groups of Dox to Fl-PMNP, consistent with previous reports.19 Mild shifts in IR spectrum and lack of new peaks formed upon Dox adsorption indicate that no significant structural changes occurred to either Dox or the NP construct. UV−vis spectra of aqueous dispersions of Fl-PMNP and Dox@ Fl-PMNP clearly show the successful loading of Fl and Dox, respectively. The absorbance of the NPs increases as Dox was loaded onto Fl-PMNP (Figure 1D), confirming that the iron oxide core is not quenching the absorbance of the fluorescent dye and drug. TGA showed ∼58% weight loss of Dox-loaded Fl-PMNPs, further confirming the successful attachment of Fl and Dox (Figure S3). Importantly, the potential utility of Dox@Fl-PMNP as MRI agents was also established by measuring its ability to enhance the T2 magnetic relaxation rate (r2*) of water in a magnetic field (Figure S4). It was shown that Dox@Fl-PMNP has excellent magnetic relaxivity (r2* = 235 mM−1·s−1), which is superior to that of Feridex (r2* = 95 mM−1·s−1), an FDA approved liver cancer imaging agent, suggesting its potential use as MRI contrast agent for potential in vivo imaging. With the Dox@Fl-PMNPs in hand, we first investigated the in vitro drug release profiles of Dox@Fl-PMNPs in PBS buffer at two different pH values at 37 °C (Figure S5). The linkage between Dox and the NPs should break readily once internalized inside the cells, with minimal premature burst release that is typically ∼20−50% in most drug delivery systems. Thus, choosing covalent linkages to attach the drug to the NP surface might not be the ideal solution. Even when the linkage is labile (i.e., hydrolyzed under acidic conditions), slow release of drugs into the media or inside the cells is typically observed and might take very long times (>24−72 h).24,43 Moreover, it is worth mentioning that the slow drug releasing particles can be degraded by lysosomes before the release of their payload. In our system, a relatively fast initial drug release over the first 4 h was observed, representing 54% Dox release at

Figure 2. (A) Dose dependent NP uptake by human breast cells as measured by flow cytometry after incubating the NPs with cells up to 24 h. The cellular binding was much higher for MDA-MB-231 compared to MCF-7, with the least uptake observed for MCF-10A. Data are presented as mean values ± standard deviations. (B) Representative flow cytometry graphs showing the death of breast cancer MDA cells before (left) and after (right) treatment with Dox@ Fl-PMNPs.

This is in agreement with previous studies where the uptake of PVP-coated iron oxide NPs increased significantly in the first 4 h and gradually slowed and reached plateau thereafter.21 Importantly, the fluorescence signal experienced 2-fold increase for MDA-MB-231 as compared to MCF-7 cells, with the least uptake observed for the normal MCF-10A. Similar phenomena have also been observed earlier.50 This can be due to the different characteristics, morphologies, and immunoprofiles D


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Figure 3. Confocal microscopy images of MDA-MB-231 cells treated with Dox@Fl-PMNPs (10 μg/mL NPs; 1.25 μg/mL Dox; 2.3 μM Dox) after 6 and 18 h. Left to right: (a) Dox (red channel), (b) Fl (green channel), (c) Hoechst (blue channel) showing positions of the nuclei, (d) overlay of the three channels. The images clearly show the internalization of NPs inside the cells, with increased uptake inside the cytoplasm and to the nucleus as time elapses.

Figure 4. Confocal microscopy images of MDA-MB-231 and MCF-7 cells after the same incubation time (6 h) with Dox-Fl-PMNPs (10 μg/mL NPs; 1.25 μg/mL Dox; 2.3 μM Dox). Overlay of Dox, fluorescein, and Hoechst channels along with transmitted light images shows that Dox@FlPMNPs are internalized more inside MDA-MB-231 compared to MCF-7, causing apoptosis to cells (typical apoptotic features such as condensation and membrane blebbing are clearly seen in the overlaid picture).

associated with the breast cancer and their respective normal cells.51 Moreover, as expected, both metastatic and normal cells showed dose dependent responses with higher uptake as the concentration of the NPs increased (results not shown). When Dox@Fl-PMNP was used, loss of the cancerous cells was observed even after short periods of incubation, pinpointing the high potency of the drug loaded NPs even at the relatively low doses employed (50 μg/mL) (Figure 2B). Next, in order to gain insights into how the NPs interacted with the cells, the accumulation of the NPs was first examined

by Prussian blue staining, which yields an intense blue color upon reaction with the magnetite core, allowing easy tracking of the particles. Incubation of MNPs with the cells led to distinctive blue color on the surface and inside the cytoplasm of the cells, while the control cells showed no blue stains (Figure S6). In order to better understand the intracellular distribution and localization of the NPs, confocal laser scanning microscopy (CLSM) and TEM studies were performed. To mimic physiological conditions, live confocal imaging with no fixation of cells was conducted. Interestingly, confocal images E


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Bioconjugate Chemistry confirmed that Dox is indeed delivered to the cell cytoplasm in relatively short periods of time in 1−6 h and to the nucleus after overnight incubation with more intense red (Dox) fluorescence observed for MDA-MB-231 as compared to MCF-7 (Figures 3 and Figure S7, respectively). In both cell lines, however, the majority of green (Fl) and red (Dox) fluorescence was found to be concentrated in the cytoplasm, and the fluorescence intensity starts to increase inside the cytoplasm reaching the nucleus, as time progresses. Interestingly, the inherent fluorescence of red Dox and green Fl seems to overlap, further confirming the π−π stacking and intercalation between the two biomolecules. Head-to-head comparison between the two cell lines at the same incubation time showed that the uptake was less for MCF-7 as compared to MDA-MB-231 at exactly the same experimental conditions, with typical apoptotic features52 clearly seen for MDA-MB-231 (Figure 4 and Figure S8). This also confirmed the cellular uptake observed using flow cytometry. On the other hand, MCF-10A showed the least florescence intensities, even after overnight incubation (Figure S9). Incubating the same cell lines with free Dox, the red fluorescence was found to be directly localized in the nucleus of cells with minimal detectable presence in the cytoplasm (Figure S10), as has been observed earlier.53,10 It is thus clear that while free Dox is internalized by passive diffusion through the cell membrane, the uptake of Dox@Fl-PMNP is directed rather by one form of endocytic trafficking mechanisms. Typically, self-organized polymeric structures or polymeric-coated nanoparticles, depending on their sizes and surface charges, chose either endocytic, phagocytic, or pinocytic pathways for cellular internalization.54 We thus hypothesize that after their uptake the Dox loaded onto Fl-PMNPs is released via the detachment of the noncovalently bound Dox from the PVPylated NPs due to the biochemical changes inside cells (mainly pH, hydrolysis, and endosomal/lysosomal hydrolytic enzymes).55 The released Dox then diffuses and traffics to the nucleus in a sustained way causing apoptosis to cells.56 In order to prove our hypothesis and further examine how the cells uptake the NPs at the subcellular level, the intracellular distribution were also evaluated by TEM imaging. Representative TEM micrographs clearly indicate that the NPs were taken up by the cells via membrane-bound vesicles, which are shuttled into the cellular cytoplasm (Figure 5). Most of the NPs were found to be either attached to the cellular membrane or located in vesicles inside the cytoplasm. Figure 5B,C showed a typical vesicle containing the NPs as it formed and moved inward. For comparison, endosomes with no NPs are visible as discrete regions within the cytoplasm (Figure 5D). It is thus clear that clusters of NPs are present in pockets created by invaginations of the cell membrane indicating NPs uptake is mediated primarily by one form of endocytosis. These endocytotic vesicles subsequently fuse with endosomes/lysosomes to hydrolyze and break down the particles. The NPs reached areas very close to the nucleus but did not cross the intact nuclear membrane. All these results indicate that the observed cytotoxic effects are dependent on the distinctive uptake of PMNPs by cells where Dox is released and then translocated to the nucleus exerting its cytotoxic action, in relatively short periods of time. We then sought to test and quantify the toxicities of the PMNPs toward the cells using the thiazolyl blue tetrazolium bromide (MTT) cell viability assay. The MTT assay is based on the capacity of the mitochondrial enzyme of viable cells to

Figure 5. (A−C) Representative transmission electron micrographs of an MDA-MB-231 cell incubated with NPs (shown in red arrows). TEM clearly shows the process of internalization, where the NPs are attached to the surface, then shuttled inside the cell cytoplasm via vesicles and packaged in endosomes near the nucleus. Note the cell membrane invaginations and vacuole formations indicative of endocytosis. (D) TEM image of control MDA-MB-231 cells without NPs. Similar phenomena were observed for MCF-7.

transform the MTT tetrazolium salt into a violet-bluish colored MTT formazan, which is proportional to the number of living cells present.57 While the unloaded Fl-PMNPs were not toxic to any of the cells, even at the high NP concentrations evaluated (100 μg/mL), the Dox@Fl-PMNPs were found to be highly toxic to MDA-MB-231 breast cancer cell line (proliferation inhibition IC50 = 17.5 ± 2.1 μg/mL NP content; equivalent to 2.5 μg/mL Dox content) and to MCF-7 (IC50 = 34 ± 3.7 μg/mL; equivalent to 4.8 μg/mL Dox content) (Figure 6). As can be seen, Dox@Fl-PMNPs showed ∼2-fold enhanced cytotoxicities against the metastatic breast cancer MDA-MB-231 in comparison to MCF-7, suggesting improved chances of chemotherapeutic response in drug-resistant metastatic cell lines. Interestingly, at the highest concentration used, up to 8-fold enhancement in potency was observed when the nontumorigenic human breast cell line MCF-10A was incubated with Dox@Fl-PMNPs at the same Dox content (IC50 = 108 ± 6.75 μg/mL NP content; equivalent to 15 μg/mL Dox content) (Figure 6). Importantly, when compared to the free drug, the potency of Dox@Fl-PMNPs toward MDA-MB-231 and MCF-7 was comparable to the potency of free Dox (IC50 = 1.7 ± 0.86 μg/mL and 6.2 ± 1.3 μg/mL, respectively) but significantly decreased for MCF-10A cells (IC50 = 2.3 ± 0.67 μg/mL free Dox). Similar IC50 values for free Dox were reported in the literature.58 Other researchers including our group illustrated that free Dox has high cytotoxicity toward those metastatic breast cells as well as to the noncancerous breast cell MCF-10A with almost similar inhibitory effects on all the cells.52,59 On the contrary, Dox@Fl-PMNPs showed lower IC50 values for the metastatic cancerous cells compared to normal breast cells, suggesting huge potential of the nanoconstructs as selective anticancer agents. It is also to be noted that the IC50 values for Dox@Fl-PMNPs are well within the range achievable in blood plasma, suggesting their clinical applicability. Taking into consideration the distinctive mechanistic cellular uptake between the free drug and drug-loaded NPs (fast diffusion vs vesicular trafficking), our results strongly F


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Figure 6. (A) Percent of viable cells upon incubation with different concentrations of NPs up to 100 μg/mL as determined by MTT cell viability assay. (B) Nonlinear regression curves of the cytotoxicity assays of Dox@Fl-PMNPs against human breast cells plotted in dose-dependent curves to calculate IC50 values. The cells were incubated with different concentrations of NPs at 37 °C for 48 h. Dox@Fl-PMNPs were found to be 2-fold more toxic to MDA-MB-231 as compared to MCF-7, with the least toxicity for MCF-10A. (C) MTT for the three different human breast cell lines treated with Dox@Fl-PMNPs showing up to 8-fold increase in potency for the metastatic cell line compared to the normal nontumorigenic cells. The experiments were carried out in triplicate, and error bars denote standard deviations.

In many cases, the cytotoxicity of drug conjugates was found to be reduced. For example, Dox-loaded NPs coated with targeting agents such as cancer-targeting peptides,65 folates,66 or RGD67 were found to be less active than free Dox. Thus, coating of a targeting ligand (i.e., antibodies, carbohydrates, peptides) on NPs does not increase the level of accumulation of the drug in the target tissue but might increase the rate of intracellular delivery.68 Furthermore, targeted NPs have slow internalization, since binding to the first encountered target cells blocks the binding of more targeted NPs.69 Maintaining the hydrodynamic size of the ligand-conjugated NPs is another challenge. Therefore, targeted NPs have not yet exhibited a significantly increased therapeutic efficacy compared to the nontargeted approach.70 In comparison, and beyond targeting expectations, it is highly advantageous that the Dox@Fl-PMNP led to much enhanced cytotoxicities. The enhanced cytotoxicity and better efficacy of Dox delivered by Fl-PMNP indicate that the prepared formulation can significantly impact the cellular distribution of the cargo and can be used to deliver agents deep inside the tumors. Penetration of Dox@Fl-PMNP in Primary Tumor Cells and Solid Tumors. In order to investigate the NP penetration

suggest that the Dox@Fl-PMNPs can be a promising platform for selective drug delivery to human breast cancers. To create therapeutic efficacy, the drug must be loaded with high efficiency, have affinity to the target, and be easily released from the NPs.60 This is a major challenge in the advanced drug delivery systems. For example, although the FDA approved the liposomal formulation of Dox (Doxil) as an effective carrier for delivering Dox, Doxil showed lower or comparable antitumor cytotoxicity vs free Dox both in vitro and in vivo.61 The major reason is the low rate of release of the drug from Doxil both in the blood circulation and in the tumor tissue. In fact, the therapeutic benefits of Doxil mostly come from the higher drug concentration reaching tumor sites through the EPR effect.62 In another example, it was found that the liposomal cisplatin formulation (SPI-077) accumulated substantially in the tumor tissue but exhibited no antitumor effect.63 Thus, high level of tumor accumulation of nanoformulations does not directly correlate to the bioavailability of the drug, which is more dependent on the rate of drug release.7,64 Although methods for targeted delivery of drugs have been investigated to enhance the drug efficacy, success has been limited. G


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Bioconjugate Chemistry into tumor tissues, we first incubated Dox@Fl-PMNPs with primary breast tumors derived directly from primary lesions. We found this is essential in order to understand the effect of NPs on the various stages of tumor progression from early stage breast cancers to late-stage progressive models. Indeed, the breast cancer cell lines in current use are typically derived from aggressive tumor metastases by pleural effusions. Thus, an important consideration that is undervalued when designing a good nanocarrier is to study its effect on primary cultures. Not only the primary tumor cells are directly isolated from the breast tumor site, but also primary cultures are more clinically relevant, particularly because most therapies are directed against them.71 To this end, primary cells were incubated with Dox@Fl-PMNPs and then subjected to live confocal imaging. CLSM images of the domelike three-dimensional (3D) clusters of the primary cells indicate almost complete penetration of the drug-loaded NPs into these 3D forming cells (Figure 7). Z-stack CSLM images are also presented in

systems, many barriers still exist.74 Unfortunately, the widely used pH-triggered release mechanism of cleavable covalent bonds is not very efficient. 5,75 Light-triggered release mechanism is also commonly considered. Nevertheless, this method is not feasible due to the poor penetration of the externally illuminated light and the necessity for tumor localization. In fact, the tumor penetration is a passive process that requires a long circulating biostable NPs to allow extravasation of the particles across the hyperpermeable tumor vessels and effective diffusion through the tumor interstitial space. The challenge remains to deliver the NPs throughout a tumor and its metastases given the limitations of spatial and temporal changes in the expression of the target. Thus, the most promising direction is to employ a rapid delivery system that either delivers NPs into the target cells by vesicle-mediated transcytosis or keeps the NPs in the microvessels of the target cell, which usually occurs within a few hours.4,76 Therefore, we anticipate that a rapid release formulation (within a few hours), as designed here, will be more desirable for the new type of delivery system. Moreover, magnetic guidance on this system is feasible by focusing an external magnetic field on the biological target after the injection of magnetically responsive nanocarrier. To gain insights into how Dox@Fl-PMNP interacts with the tumor tissue, we incubated breast cancer biopsies with Dox@ Fl-PMNPs or free Dox for 1 week at the same subtoxic concentrations. Dissected breast tumor biopsies were approximately 1 mm3 in size and were cultured to mimic oxygen, nutrient, and energy gradients similar to those found in vivo. Zstack confocal microscopy images starting from top to bottom of patient biopsy sample exposed to Dox@Fl-PMNPs were acquired. As shown in Figure 8, extensive distribution of the Dox fluorescence through the tumor from top to bottom, which overlaid to certain extent with the green fluorescence, was observed. An intensity profile of the three overlaid fluorescence is also shown in Figure 8. The 3D reconstructed images showed areas of extensive red Dox deeper inside the tissue (as shown in arrows), while no green Fl was seen, signaling significant intracellular Dox release and death to the cells inside the tumor (Figure 9). More 3D views of the tissue can be seen in Figure S12. It is possible that transcytosis of the NPs after delivery occurred, similar to previous observations,77 or the NPs penetrate the tissue to a certain level releasing the drug internally. Nevertheless, from our results, it is clear that Dox@ Fl-PMNPs exhibited acceptable penetration (∼15%) where Dox was found ∼150 μm deep into the tumor. In comparison, the amount of red fluorescence penetration was clearly much lower for free Dox (Figure S13 and Figure S14), confirming the recognized limited penetration of free drugs through tumor tissue and hence the resistance of solid tumors to chemotherapy.73 All these observations confirm the potential of Dox@ Fl-PMNPs as effective drug delivery carriers that can access the interior of the tumor, significantly improving tumor penetration ability of drugs. Importantly, this designed nanoformulation is promising to induce enhanced therapeutic activity in breast cancer patients and may further allow physicians to magnetically image the cells exposed to PMNPs. This can potentially open new opportunities for in vivo combined therapeutic imaging and hyperthermia.

Figure 7. 3D reconstruction CLSM images of primary breast tumor cells treated with Dox@Fl-PMNPs: (a) Dox channel (red color), (b) Fl channel (green color), (c) Hoechst channel (blue color), (d) overlay of Hoechst, Fl, and Dox channels. Imaging data clearly indicate almost complete penetration of drug-loaded NPs in the domelike 3D cells, killing the cells effectively.

Figures S11. These promising results corroborated the successful penetration of Dox@Fl-PMNPs into even 3D primary tumor cells, which gave us great confidence to further test the penetration into tumor tissues. Around 90% of promising preclinical drugs fail to result in efficacious human trials. Abnormal tumor architecture, inadequate drug accumulation, and tumor drug resistance are tightly linked together and make drug penetration extremely difficult throughout the tumor microenvironment.72 If anticancer drugs are unable to access and penetrate all the cells within a tumor, then their therapeutic effectiveness will be hampered, no matter what is their actual potency. Given the uniform results, showing limited distribution of Dox in solid tumors,73 it is critical to develop new nanocarriers that can improve the delivery into solid tumors and hence enhance its efficacy. Many trials to enhance the efficacy of nanoformulations have been developed by constructing NPs that respond to heterogeneity of the tumor microenvironment (i.e., low pH, redox, enzyme, etc.) or to external forces (i.e., electric pulses, magnetic field, heat, light, etc.). Despite the existence of different strategies for stimuli-responsive triggered drug release

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CONCLUSION We developed a chemotherapeutic nanoformulation loaded with anticancer drug as a promising drug carrier to different H


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Figure 8. Z-stack image gallery starting from top to bottom of patient tumor biopsy sample exposed to Dox@Fl-PMNPs for 7 days. To the left is a diagrammatic representation illustrating the penetration of Dox@Fl-PMNPs inside the tissue containing extracellular matrix (ECM) and the other cellular components.72 Intensity profile of the three overlaid fluorescence. Image shows penetration of Dox up to 150 μm deep into the tumor tissue.

confocal, and electron microscopy. While the Fl-PMNPs were highly biocompatible, the Dox@Fl-PMNPs were found to be highly potent to the different metastatic breast cancer cells, with considerably less toxicity toward the normal nontumorigenic breast cells. While free Dox is concurrently toxic to the three cell lines, Dox@Fl-PMNPs showed lower IC50 values for the metastatic cancerous cells compared to normal breast cells, suggesting huge potentials as selective anticancer agents for breast cancer therapy. Mechanistically, the Dox@Fl-PMNPs were found to be endocytosed and shuttled into vesicles inside the cytoplasm, where Dox is then released and translocated to the nucleus exerting its cytotoxic action, in relatively short periods of time. Importantly, Dox@Fl-PMNPs were also able to effectively penetrate 3D forming primary tumor cells and patient tumor biopsies. While many prior studies have focused on targeting ligands to specific receptors to improve efficacies, we discovered that even with nonreceptor targeted customized delivery system enhanced toxic responses can be achieved.

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Figure 9. 3D reconstruction of patient biopsy tumor tissue exposed to Dox@Fl-PMNPs for 7 days. Images show almost homogeneous distribution of Dox (red) around the tissue, whereas Fl-PMNPs (green) are rather heterogeneous, suggesting that the NPs may have excreted out (transocytosed) after delivering their payload.

EXPERIMENTAL SECTION Materials and Methods. Unless otherwise indicated, all chemicals and solvents were obtained from commercial suppliers and used as supplied without further purification. Iron(III) chloride hexahydrate (FeCl3·6H2O), iron(II) chloride tetrahydrate (FeCl2·4H2O), potassium ferrocyanide trihydrate [K4Fe(CN)6], fluorescein (Fl), and doxorubicin (Dox) were all purchased from UFC Biotechnology. Poly-N-vinylpyrrolidone

types of breast cancer cells, primary tumor cells, and solid tumors. With its superior properties, the Dox@Fl-PMNPs enabled the monitoring of NP uptake by fluorescence imaging, I


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NH4OH (∼1 mL) was then slowly added, and stirring was continued for 3 h. The nanoparticle suspension was then isolated via centrifugation (4500 rpm, 10 min), washed repeatedly with ethanol and water until no fluorescence was detected in the supernatant, and finally redispersed in water to form remarkably stable aqueous colloidal dispersions of FlPMNPs (10 mg/mL). Alternatively, the suspension can be dialyzed against water until no fluorescence is detected. Next, 4 mL of aqueous dispersion of Fl-PMNPs (2 mg/mL) and 0.5 mL of Dox solution (1 mg/mL) containing a few drops of NaOH (0.1 M) were gently shaken on a rotary shaker at 37 °C for 36 h to enable maximal Dox loading. The pH of the solution was then adjusted from basic to neutral by the addition of 0.1 M aqueous HCl solution. The NP dispersion was then isolated via centrifugation (4500 rpm, 20 min), washed repeatedly with water until no Dox was detected in the supernatant, and finally redispersed in water to form stable aqueous suspensions of Dox@Fl-PMNPs. The aqueous suspension was further purified by dialysis against distilled water to remove any residual Dox. The percentage of Dox on NPs was determined by UV−vis spectroscopy. The absorbance of the residual Dox in the supernatant was measured (λmax = 490 nm), and the percentage of Dox loading (w/w%) was then quantified. The loading efficiency was calculated as

(PVP) (average MW of 58 000) was purchased from Alfa Aesar. MDA-MB-231, MCF-7, and MCF-10A human breast cell lines were purchased from American Type Culture Collection (ATCC). Dulbecco’s phosphate buffered saline (DPBS), phosphate buffered saline (PBS), advanced Dulbecco’s modified Eagle medium (DMEM), phenol-red free DMEM, fetal bovine serum (FBS), Hoechst 33342 stain, L-glutamine, and penicillin−streptomycin (Pen-Strep) were all purchased from Invitrogen or UFC Biotechnology. MTT (thiazolyl blue tetrazolium bromide) was purchased from Bioworld, USA. All reactions for the NP syntheses were carried out under an argon atmosphere. 0.2 μm filtered deionized water was used for the synthesis of the nanoparticles. For the cancerous cell lines, MCF7 and MDA-MB-231 were grown in advanced DMEM containing 10% FBS, L-glutamine, and 1% antibiotics (PenStrep). MCF10A cells were cultured in Advanced DMEM/ Ham’s F-12+Glutamax-1 (GIBCO, USA) supplemented with 100 ng/mL cholera toxin, 20 ng/mL epidermal growth factor (EGF), 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, and 5% equine serum. All of the growth factors were purchased from Sigma (St. Louis, MO, USA). Early passages of MCF10A cells were used in all the experiments. Cells were cultured for at least 24 h before conducting the experiments. All experiments were conducted in triplicate, and mean averages were plotted. Tumor biopsies and surgical sections were collected after examination by a certified pathologist, prepared for analysis on the same day, and cultured in advanced DMEM. Tissues were collected at the time of surgery from consenting patients at King Abdulaziz Medical City, National Guard Hospital, under approval from the institutional review board. Primary tumor cells were collected from tumor surgical sections cultured for several weeks in advanced DMEM. These cells make domelike 3D structures with multilayer nature of these domes near confluency. Tumor biopsies and primary tumor cells were transferred into 8-well dishes (Nunc Technologies) prior to NP exposure, and cell culture medium was replaced by phenol-red free DMEM during treatment. FTIR spectra (400−4000 cm−1) were recorded as KBr pellets using Shimadzu IRAffinity-1. TGA was carried out on a PerkinElmer TGA 4000 equipment, and the samples were burned under nitrogen at a constant heating rate of 10 °C/min from 35 to 700 °C. DLS measurements were assessed on Malvern Zetasizer Nano ZS instrument. TEM images were collected on a JEOL-JEM 1230 operating at 100 kV using Gatan camera with DigitalMicrograph imaging software. Nanoparticle samples were prepared by depositing 5 μL of the particle dispersion onto 400 mesh formvar/carbonsupported copper grid. The suspension was then allowed to air-dry overnight before images were taken. For the nanoparticle-cell specimens, see the detailed TEM procedure below. Flow cytometry was conducted on Beckton Dickinson FACSCANTOII. Confocal images were visualized using inverted Zeiss LSM 780 multiphoton laser scanning confocal microscope equipped with 20× and 40× (oil immersion) objectives and axiocam cameras. Optical images were taken on Zeiss AxioImager inverted microscope. Preparation of Dox@Fl-PMNPs. PVP (16 mg) and fluorescein (varied between 2 and 30 mg, 13 mg preferable) were dissolved in 1 mL of water/DMF (10:1) in the presence of a few drops of 1 M HCl solution (pH = 4) and stirred for 30 min at 37 °C under argon. To this solution, FeCl3·6H2O (30 mg, 0.11 mmol) in water (1 mL) was added, followed by addition of aqueous FeCl2·4H2O (11 mg, 0.055 mmol).

loading efficiency =

Wl × 100 W0

where Wl is the amount of Dox loaded onto NPs and W0 is the amount of Dox in the initial solution. The amount of Dox adsorbed onto NPs was calculated from the difference between the initial Dox concentration and the Dox concentration in the supernatant. In Vitro Drug Release. In a typical study, a certain amount of dried drug-loaded NPs (10 mg) was suspended in 1 mL of PBS buffer at two different pH values (pH = 7.4 and pH = 4.5) and gently rotated at 37 °C in an oven provided with a precise control in the temperature within 0.01 °C. The concentration of Dox in the supernatant was determined at fixed time intervals by UV−vis spectroscopy. At specific time points, supernatant aliquots obtained by centrifugation were taken from each tube, measured, and returned to their respective tubes after UV−vis measurements. The drug concentration could be directly calculated from the measured absorbance. The percentages of released drug were calculated as the ratio of released Dox to the initial concentration of Dox at t0. Triplicate aliquots were run for each time interval, and average values were plotted. Cell Viability Assay. Cell viability of breast cancer cells exposed to NPs was determined using MTT assay. The cell lines were seeded in a 96-well plate at a density of 5 × 105 cells/ well and incubated in 95%/5% humidified air/CO2 at 37 °C. After 24 h, the medium was removed and fresh phenol-red free DMEM containing 0.5% FBS was added to the cells. Cells were then treated with various concentrations of free Dox, Dox@FlPMNPs, or Fl-PMNPs in 200 μL of supplemented DMEM. After 48 h of incubation, the medium was removed and the cells were washed with PBS. Cell viability was then determined using the MTT viability assay following the manufacturer’s protocol. Briefly, 20 μL of MTT reagent (5 mg/mL) was added to each well and kept for 4 h at 37 °C in the incubator. The supernatant was then removed, and the MTT formazan was dissolved in dimethyl sulfoxide (DMSO). The absorbance was J



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measured on iMark microplate absorbance reader at 590 nm. The percentage of viable cells was calculated as the ratio of the absorbance of the treated group divided by the absorbance of the control group multiplied by 100. The absorbance from the untreated control cells was set as 100% viable. IC50 values were calculated from dose−response curves generated using a polynomial dose−response approximation. Prussian Blue Staining. Cancer cells (5 × 104 cells/well) were allowed to attach overnight in a well plate at 37 °C and 5% CO2. The medium was removed. Fl-PMNPs (10 μg/mL) were added and incubated with the cells for 6 h, after which the supernatant was removed and the cells were washed three times with PBS. The cells were fixed with 10% formalin (0.5 mL/ well) for 5 min and then washed with PBS twice. To each well 0.5 mL of a 1:1 mixture of 4% potassium ferrocyanide (II) trihydrate and 4% HCl solution (in PBS) was added, and the cells were incubated in the dark at 37 °C for 30 min. The staining solution was removed, and the cells were washed with PBS and counterstained with nuclear fast red. The supernatant was then removed, and the cells were washed to remove any remaining staining solution. Optical images were then taken on Zeiss AxioImager inverted microscope. Monitoring Penetration of NPs by Live Confocal Imaging. Cells were incubated in 8-well dish (Thermo-fisher Scientific) for 24 h prior to PMNPs exposure. After removing the supernatant, cells were then exposed to Fl-PMNPs (10 μg/ mL), Dox@Fl-PMNPs (10 μg/mL NPs; 1.25 μg/mL Dox), or the equivalent amount of free Dox. The cells were incubated for different periods of time. The supernatant was then removed, cells were washed, and Hoechst 33342 stain was added 5 min before microscopic visualization. To mimic physiological conditions, no fixation of cells was conducted. Fluorescence Activated Cell Sorting (FACS). Breast cancer cells (2 × 106 cells) were seeded on 6 cm diameter culture dishes, incubated for 24 h, and then exposed to FlPMNPs (same final concentrations used for previous experiments) for different periods of time. Representative flow cytometry graphs after treatment with Dox@Fl-PMNPs (50 μg/mL) were also recorded. Both live and dead cells were collected, pelleted, washed with PBS (3×), and then transferred to FACS tubes for analysis. Flow cytometry measurements were then assessed. Triplicate experiments were conducted. Transmission Electron Imaging of NP-Cell Specimens. Cells were first treated with the NPs (10 μg/mL) as described above. The specimens were then processed for TEM by the following method: the specimen was fixed in 3% glutaraldehyde in 0.1 M PBS (pH = 7.4) for more than 3 h. After washing in the same buffer, this was postfixed in 1% OsO4 in 0.1 M PBS (pH = 7.4) for 1 h, followed by PBS washing. The specimen was then dehydrated in a series of acetone solutions and infiltrated in acetone/resin (1:1) for 1 h and then with acetone/ resin (1:2) for more than 3 h. The specimen was then embedded in epoxy resin (Araldite) and placed in an oven at 80 °C overnight to polymerize. Ultrathin sections were obtained with Ultra microtome (Leica EM UC6), mounted on copper grids, and stained for contrast with heavy metal stains (uranyl acetate and lead citrate). TEM images were then collected on a JEOL-JEM 1230 operating at 100 kV using Gatan camera with DigitalMicrograph imaging software.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00257. UV−vis spectra, ζ potential, TGA, relaxation rates, drug release profiles, and confocal images (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. sa. Author Contributions §

K.H.B. and R.A. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by KAIMRC through Grant RC13/204/ R. The authors acknowledge the continuous support by KSAUHS, NGHA, and KAIMRC. The authors also thank Dr. Ibraheem Bushnak, Dr. Rabih AlKaysi, Mutaz Al-Ghamdi (UV−vis), Hajar Al-Zahrani (cell culture), and Thadeo Trivilegio (flow cytometry) for their assistance and help in the study as indicated.

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DEDICATION We dedicate this work to Prof. Xuefei Huang. REFERENCES

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