Red Blood Cells-Derived Vesicles for Delivery of Lipophilic Drug

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 22141−22151

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Red Blood Cells-Derived Vesicles for Delivery of Lipophilic Drug Camptothecin Sahil Malhotra,† Shweta Dumoga,† Parul Sirohi,‡ and Neetu Singh*,†,§ †

Centre for Biomedical Engineering and ‡Department of Chemistry, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi 110016, India § Biomedical Engineering Unit, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

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S Supporting Information *

ABSTRACT: Recently, cell membrane-derived nanoparticles, particularly of RBCs, have been explored for delivery of hydrophilic solutes of varied size and complexities. So far, these naturally derived nanoparticles show a significant overlap with liposomes in terms of stability, solute encapsulation, and release. Unlike hydrophilic molecules, which are loaded inside the aqueous core, hydrophobic moieties largely partition inside the lipophilic shell, hence fate of these nanocarriers may be different. Since vesicles have more complex membrane architecture (due to natural lipids and additional proteins and glycoproteins), ease of loading hydrophobic drug, its release pattern, and overall particle stability cannot be compared to those of synthetic lipid-based carriers. Therefore, we derived nanovesicles (NVEs) from RBC membrane, loaded with hydrophobic drug camptothecin (CPT) and labeled noncovalently with amphiphilic fluorophore (CM-DiI). Although both CPT and CMDiI are known to partition inside the membrane, the overall stability of NVEs and composition of membrane proteins, particularly CD47, “marker of self”, did not change. Additionally, the developed NVEs were found to be nonphagocytic even in the presence of serum and showed minimal stimulation of macrophages to release cytokines. Further, this system showed slow release but strong retention of CPT and CM-DiI, respectively, over 24 h, hence appropriate for theranostic applications. Also, NVEs were internalized by lung carcinoma cells and possessed slightly higher toxicity than free CPT. When injected intravenously in balb/c mice, these nanovesicles showed higher retention in blood over 48 h and insignificant accumulation in vital organs like heart and kidneys, thus suggesting its potential for in vivo application. We believe that this system has superior stealth and comparable physicochemical properties to synthetic lipid-based nanocarriers; hence, it can be further developed as personalized medicine. KEYWORDS: RBC-derived nanovesicles, camptothecin, CM-DiI, theranostic, lung carcinoma clearance mechanism toward synthetic NPs.7,8 However, the process of RES clearance can be slowed if surface of the nanoparticles is decorated with hydrophilic polymers like poly(ethylene glycol) (PEG).9,10 It inhibits opsonization, key process responsible for recognition of nanoparticles as immunogenic to macrophages.11 Consequently, PEG coating was fundamental for providing “stealth” to any type of nanoparticles like liposomes and polymeric micelles.4 How-

1. INTRODUCTION In recent years, nanotechnology has improved therapeutic efficacy of pharmaceutical products and mitigated its side effects.1−5 Nanoparticles (NPs) can be tailored to load drugs with different chemical entities, and ease of their surface functionalization enables tissue-specific drug delivery.6 Their optimal size range not only prevents rapid renal clearance of the drug, but also does not obstruct the submicron-sized blood vessels. Since their size and surface morphology (particularly those of liposomes) mimic to a great extent with pathogens like bacteria and virus, our innate immune system (particularly reticuloendothelial system (RES)) can evoke a similar © 2019 American Chemical Society

Received: March 18, 2019 Accepted: May 31, 2019 Published: May 31, 2019 22141

DOI: 10.1021/acsami.9b04827 ACS Appl. Mater. Interfaces 2019, 11, 22141−22151

Research Article

ACS Applied Materials & Interfaces Scheme 1. Formulation of Vesicles (NVE) from RBCs

Figure 1. (a) Transmission electron micrograph of NVEs, scale bar = 200 nm; (b) size and (c) ζ-potential of RBCs, ghosts, unloaded NVEs (NVE) and CM-CPT-loaded NVEs (CMNVECPT) obtained through DLS; (d) stabilities of NVE and CMNVECPT at 37 °C in phosphate-buffered saline (PBS) and fetal bovine serum (FBS) at 0 and 24 h, respectively; the error bars include the standard error bars. (e) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation of membrane proteins of NVE and CMNVECPT; (f) size measurement of NVEs tagged with primary CD47 antibody and Alexa Fluor 568-labeled secondary antibody.

easier to utilize its plasma membrane to derive nanovesicles (NVEs). These particles have been explored either as an alternative to PEG for coating other nanoparticles or directly to encapsulate hydrophilic drugs. For example, Zhang et al. showed that RBC membrane-coated PLGA NPs show similar stability but prolonged system circulation compared to PEGcoated NPs.24 The membrane sidedness of RBCs remains conserved during the top-down approach of NPs synthesis, and overall protein content also remains unchanged, of particular interest is the conservation of CD47, a “marker of self”.27 Another report from the same group demonstrated that doxorubicin hydrochloride can be loaded inside the vesicles via ammonium sulfate gradient, much in a similar way it can be loaded in liposomes.28 Similarly, these particles were found to be comparable to conventional liposomes in the context of solute encapsulation, release pattern, and ease of conjugating antibodies.25 Therefore, while these reports suggest the ease of loading hydrophilic drugs inside the aqueous core of vesicle, there are insufficient data to conclude the usefulness of its complex lipid bilayer in delivering hydrophobic drugs. NVE membrane, unlike that of liposomes, is heterogeneous in chemical composition and fluidity. It is made up of domains where proteins and glycoproteins are embedded inside the lipid bilayers, where such architecture is essential for stability

ever, later it was found that, although PEG decelerates opsonization of proteins on NPs surface, it can elicit another type of nonspecific immune response like CARPA and generation of anti-PEG antibodies in some individuals.7,12−14 The severity of such immune response in vivo cannot be predicted, which can limit the overall dosage of the drug, thus reducing its therapeutic potential.7 In a quest of delivery vehicles with less immunogenicity and optimum physicochemical properties similar to pegylated NPs, researchers have explored the concept of personalized medicine. This concept manifests in either formulating nanoparticles from host’s own cells, loading them with drugs of choice, or coating the synthetic NPs with cellular membrane and reinjecting these particles into individual’s body. Exosomes15−21 and cell membrane-derived vesicles22−26 are examples that have core shell morphology with lipid bilayer shell and an aqueous core. Additionally, these vesicles can be superior in stealth properties compared to their synthetic analogues as they inherit surface proteins and antigens from host’s cells and hence, these nanoparticles can be akin to individual’s body. In this regard, cell membrane-derived nanoparticles, particularly those derived from red blood cells (RBCs), are important.24 RBCs can be easily extracted from peripheral blood, and since they lack cellular organelles, it is 22142


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increasing the overall size of NVEs. However, it was easier to further reduce its size (189.3 ± 3 nm) by giving two extra cycles of extrusion. ζ-Potentials (Figure 1c) of NVE and CM NVECPT were determined to be −7.87 ± 0.32 and −8.24 ± 0.92 mV, respectively, less negative than those of RBCs −13.4 ± 0.17 mV and ghosts −13.9 ± 1.48 mV. While the ζ-potential of RBCs is similar to that reported elsewere,33 negative values of all types of particles are likely due to their similar surface compositions.24 Interestingly, the ζ-potential of vesicles was unchanged when both CM-DiI and CPT were partitioned inside the membrane. This is because the ζ-potential of cellular membranes depends on the surface charge,33 which is unlikely to be changed on incorporation of electrically neutral molecules of CPT. After characterizing drug-dye-loaded vesicles for their size and surface charge, it is important to examine their stability in biological milieu. Size of the particles in PBS and 10% FBS (Figure 1d) was determined by DLS over 24 h. This time frame is critical to ensure stability of particles until complete release of drug takes place. It was found that hydrodynamic diameter of both the NPs remain unchanged even after 24 h of incubation, suggesting that partition of hydrophobic CPT and amphiphilic CM-DiI inside the vesicles membrane had insignificant effect on its stability. This observation suggests that vesicles membrane is flexible enough to provide room for hydrophobic but structurally rigid moieties, without losing the overall stability of the particles. Next, the gross change in membrane protein content, which might be possible on incorporation of foreign molecules, was examined by SDSPAGE. Not much difference in protein bands of NVEs and NVEs co-loaded with CPT and CM-DiI was evident (Figure 1e). To further confirm if the nanovesicles retain CD47, which gives them the stealth property,34 the NVEs were incubated with mouse anti-CD47 primary antibody and labeling it with Alexa Fluor 562-labeled secondary antibody. The samples were analyzed through particle size analyzer, which can detect size distribution of nanoparticles with or without fluorescence mode. A significant overlap of vesicle sizes (Figure 1f) when measured with and without fluorescence clearly suggested that most of the vesicles had fluorescence from the secondary antibody, indicating the presence of CD47. From the above experiments, it can be concluded that stability and basic structural features of cellular membrane-derived nanoparticles remain conserved on incorporating model hydrophobic drug camptothecin. Next, the presence of CM-DiI on drug and dye co-loaded vesicles (CMNVECPT) was analyzed by fluorescence microscopy and spectroscopy. Vesicles were seen as tiny red spots (Figure S1c) when excited by green light, due to excitation of CM-DiI. On exciting the samples at 530 nm, a blue shift in emission maxima of CM-DiI (Figure S1d) was observed when its solvent polarity was changed (λem = 575 nm in ethanol; λem = 596 nm in PBS). Interestingly, the λem values of CMNVECPT taken in PBS lie at 570 nm, much closer to that of free CM-DiI in ethanol. Such similarity in fluorescence spectra of CM NVECPT and CM-DiI in ethanol reveals that dye molecules in NVEs experience microenvironment of similar polarity; hence, dye is largely present inside the lipophilic membrane. However, the fluorescence spectra of CPT have lower sensitivity toward its solvent polarity.31 Therefore, it is not possible to deduce CPT localization inside the vesicles based on fluorescence spectra.

of vesicles. Thus, it is crucial to sustain intricate balance of noncovalent forces between membrane lipids and proteins on the incorporation of hydrophobic drugs. Exceptionally strong affinity of membrane lipids for hydrophobic drug molecules may increase protein−protein interaction, thus precipitating proteins inside the membrane, leading to particles aggregation. Similarly, microenvironment of NVEs membrane should be favorable enough to allow maximal retainment of drug but not as hydrophobic to give a poor release pattern. Thus, it is important to study interaction of cell membrane-derived vesicles with hydrophobic drugs from the standpoint of NPs stability and their ability to effectively load and release the drug. Here, we have used camptothecin (CPT) as a model hydrophobic drug. Free CPT is insoluble in water and undergoes pH-dependent hydrolysis to less potent carboxylate ion.29,30 It is easy to detect and quantify CPT based on its fluorescence in the blue region when excited by long ultraviolet light.31 For tracking NVEs inside the cells, particles were noncovalently labeled with an amphiphilic dye, CM-DiI, which is excited by green light, hence does not interfere in the detection of CPT. We describe here simultaneous loading of CPT and fluorophore into nanoparticles derived from RBCs. These vesicles were characterized for their physical properties like size, ζ-potential, stability, and surface protein content. Before studying its theranostic applications, a detailed in vitro study on biocompatibility of unloaded vesicles was performed. Having confirmed its compatibility, loading of CPT inside the vesicles and their release pattern were evaluated. As CPT derivatives are approved for treating lung cancer,32 experiments pertaining to its uptake and cytotoxicity of NVEs were carried out with lung carcinoma A549 cell lines. Later, this NVEs system was explored to evaluate the pharmacokinetic and biodistribution profile in balb/c mice model for in vivo application.

2. RESULTS AND DISCUSSION 2.1. Nanovesicle Characterization. NPs were derived from RBCs using the top-down approach, as given in Scheme 1. The resulting vesicles were characterized for their size and morphology through transmission electron microscopy (TEM) after staining with 1% uranyl acetate. The TEM image (Figure 1a) depicts core shell morphology of the vesicles, with overall size of approximately 150 nm and lipid bilayer thickness of 9 nm. Previously, vesicles were also derived from less tedious probe sonication method, but on the contrary, it gave more fragmentation, as seen in the TEM image (Figure S1a,b). Also, better control and reproducibility in size distribution of the nanoparticles was obtained through extrusion. Thus, in contrast to sonication, extrusion was opted for obtaining nanovesicles from ghosts. The size was also analyzed by dynamic light scattering (DLS) (Figure 1b). The hydrodynamic diameter of the unloaded vesicles (NVE) was 207.5 ± 0.3 nm with PDI range 0.1−0.2. Further, particle concentration was also measured by Nanoparticle Tracking Analysis, Malvern, U.K. It was estimated that 1 μL/mL of RBCs yielded 5.34 × 107 nanovesicles per mL of suspension. Additionally, fluorophore (CM-DiI)-labeled vesicles co-loaded with drug (CPT) were synthesized. The resulting vesicles (CMNVECPT) had slightly higher size than unloaded NVEs (250 nm, data not shown) when same number of extrusion cycles were given. This increment in size is expected since CPT, being hydrophobic, will partition largely inside the shell, thus 22143


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Figure 2. (a) Percent hemolysis of NVE with respect to 1% TX-100 as positive control (PC); (b) percent cell viability of A549 cells incubated with free NVEs and determined through MTT assay; and (c) percent bovine serum albumin (BSA) adsorption of RBCs, ghosts, and NVEs at 1, 6, and 12 h, respectively. The error bars include the standard error bars.

Figure 3. (a) Uptake of CMNVE by macrophages evaluated by flow cytometry at 4 h. Concentrations of (b) TNF α and (c) IL-6 in cell culture supernatant of macrophages treated with NVEs at 12, 24, and 48 h.

surface modification with PEG cannot prevent opsonization absolutely.38 Albumins have predominately higher concentration in plasma and thus form the bulk of protein corona formed on nanoparticles surface.40 Therefore, the extent to which bovine serum albumin (BSA) adsorbs on NVEs surface was first studied and compared to intact RBCs and resealed erythrocytes. After 1, 6, and 12 h incubation of nanovesicles with 1 mg/mL BSA solution, the percent protein adsorption was evaluated by bicinchoninic assay (BCA). Results (Figure 2c) show that similar amount of BSA (20−30%) was adsorbed onto RBCs, resealed erythrocytes, and NVEs. Although initially more BSA was adsorbed onto ghosts and NVEs (28−30%) compared to rbcs (20%), this difference was ruled out after 12 h, similar to experiments shown earlier.41 This is because surface area-to-volume ratio of ghosts and NVEs is larger compared to rbcs and hence relatively more protein adsorption. Next, we studied the phagocytic activity of macrophages toward NVEs both in the presence and absence of human serum. CM-DiI-labeled NVEs (CMNVE) were incubated with macrophages for specific time and cells were fixed and analyzed through flow cytometry. Results (Figure 3a) show that no significant shift in fluorescence intensity of CM NVE-treated cell population was evident after 1 h (Figure S1e) and 4 h (Figure 3a) when cells were incubated at 37 °C. This inactivity of NVEs toward phagocytosis could be due to insignificant opsonization of NVEs and conservation of CD47 on its surface. Thus, it can be concluded that NVEs, when administered in vivo, may have the capability of circulating for longer time and remain camouflaged with host’s immune system. 2.2.3. In Vitro Cytokine Release Assay. Nanoparticles can also have immunomodulatory effects, which can either suppress or activate the immune system.42 In both ways, it can have detrimental effects on the immune function and overall therapeutic potential of nanoformulation. According to McNeil et al.,35 there are in vitro assays, which are reliable in predicting in vivo immunotoxicological fate of nanoparticles. Therefore, in vitro experiments were carried out to study the interaction of NVEs with components of immune system.

Thus, all of the above observation states that NVEs can incorporate CPT and CM-DiI simultaneously, can be characterized, and can form stable systems and, in addition, retain basic features of the parent cells, from which they were derived. 2.2. Biocompatibility of Nanovesicles. 2.2.1. Hemoand Cytocompatibility. Mostly, nanoformulations are administered intravenously, which directly interact with rbcs. Many synthetic nanoformulations have the ability to cause hemolysis, in spite of having optimum in vitro properties.35−37 In this regard, nanoparticles derived from RBCs can be used safely since they have similar membrane compositions. NVEs, in the concentration range 5−25 μL/mL (expressed as volume of RBCs/mL of suspension from which they were derived), were incubated with RBCs for 2 h according to a previously defined protocol, and absorbance of hemoglobin released in the supernatant was determined at 540 nm. All NVE samples showed less than 1% hemolysis (Figure 2a) compared to positive-control Triton X (TX-100). This value is less than the accepted hemolysis rate of 5%, and hence NVEs can be safely injected as far as its hemocompatibility is concerned. Another study to evaluate cytocompatibility of NVEs (Figure 2b) was carried out with lung carcinoma A549 cells by incubating them with varied concentrations of NVEs for 48 h. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to elucidate percent cell viability of cells with respect to untreated cells. Results reveal that percent cell viability of all treated cells was similar to that of untreated cells, even for the highest concentration of NVEs (25 μL/mL) used in the experiments. This can be anticipated based on the fact that nanoparticles used here are made up of cellular components, which produce nontoxic degradation products. Thus, NVEs can be considered as both hemo and cytocompatible. 2.2.2. Protein Adsorption and Phagocytosis. It is now widely accepted that plasma proteins influence the biodistribution of nanoparticles and may accelerate the rate of particles clearance from blood circulation.38,39 It is evident that nearly every type of nanoparticles tend to adsorb proteins, even 22144


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Figure 4. (a) Percent loading of CPT inside the vesicles determined through (b) percent release profile of CPT from CMNVECPT and (c) retention of CM-DiI inside CMNVECPT. The error bars are the standard error bars.

lipophilic lactone ring, which interacts with lipid bilayer of rbcs, thus helping in its binding. 2.3.2. Release Profile of CMNVECPT. Release of both drug and dye from NVEs was investigated via dialysis. To facilitate the passive diffusion of solutes across the dialysis membrane, a membrane with MWCO 8−10 kDa, at least 8−10-fold higher than the molecular weight of solute molecules, was selected. Samples were loaded into a dialysis bag, and at specific time intervals, suspension was taken out and its total drug and dye contents were quantified through fluorescence. It was found that free CPT was released readily (Figure S1f), with more than 90% release within 2 h; on the contrary, its nanoformulation (CMNVECPT) showed only 5.5% release of CPT (Figure 4b) within 1 h, 30% in 2 h, and approximately 50 and 80% of the drug was released in 6 and 24 h, respectively. Its release pattern is similar to CPT-loaded multilamellar liposomes reported earlier by Giovanella et al.,43 which showed nearly 50% release of CPT in the first 6 h. Thus, NVEs mimic liposomes in their release pattern, in spite of additional presence of membrane proteins on the vesicles surface. Burst release of CPT observed with NVEs and liposomes43 is probably due to intercalation of hydrophobic drug inside the lipid bilayer and partly due to diffusion barrier for the drug localized inside the core. Interestingly, while free CM-DiI showed 45 and 70% release in 2 and 24 h, respectively, strong retention of CM-DiI was observed inside CMNVECPT over 24 h (Figure 4c). While, initially, vesicles solubilized both CPT and CM-DiI effectively, their membrane had differential affinity toward both. We hypothesize that, although both are lipophilic, strong retention of the fluorophore with sustained release of CPT is likely due to their different molecular structures. CMDiI has somewhat amphiphilic character, with hydocarbon chain and polar indole ring. Its incorporation inside the bilayer is similar to addition of another “lipid like” moiety, which may preserve the short-range order among the membrane lipids. On the contrary, CPT having conformationally rigid rings might be a “less fit” inside the bilayer structure, and hence, its release could restore the local distortions otherwise made, in the shortrange order of the lipids. Such kind of biocompatible system might prove ideal for theranostic applications in vivo. 2.3.3. Uptake of CM-DiI-Loaded Vesicles by Lung Carcinoma Cells. For studying the internalization of fluorophore-conjugated NVEs (CMNVE) by lung carcinoma cells, vesicles were incubated with cells for 4 h. To rule out any

Proinflammatory cytokines are produced by immune cells in the case of their interaction with foreign particles, which are key mediators and hence biomarkers of immune response.42 Thus, we analyzed the potential of NVEs to stimulate THP-1 cell-derived macrophages by measuring levels of proinflammatory cytokines TNF α and IL-6 in cell culture supernatant. Baseline concentration of TNF α (untreated cells, blank) was found to be in the range of 20−40 pg/mL between 12 and 48 h (Figure 3b). On the contrary, lipopolysaccharide (LPS) (positive control) induced maximum production of TNF α at 12 h (285 ± 110 pg/mL), while NVE-exposed cells showed only marginal increase in its concentration (20−50 pg/mL range). For IL-6 (Figure 3c), baseline concentration was determined to be in the range of 150−200 pg/mL, whereas for LPS- and NVE-treated cells, they were found to be in the range of 1000−1500 and 150−300 pg/mL, respectively. Concentration of NVEs used in this experiment was 25 μL/mL, which is expressed as volume of RBCs per mL of suspension, from which NVEs were derived. This value was much higher than the RBCs count used for loading the highest concentration of CPT. Thus, it can be inferred that the interaction of NVEs with macrophages showed only minimal increase in TNF α and IL-6 expression even with high concentrations of NVEs. In conclusion, NVEs were found to show acceptable hemo and cytocompatibilities, virtually insignificant phagocytosis by macrophages, and minimal elevation of proinflammatory cytokines in vitro. Hence, it was justified to further extend our work to study theranostic applications of NVEs using CPT as a model drug. 2.3. Theranostic Properties of NVEs. 2.3.1. Loading Efficiency of CPT-Loaded NVEs. CPT suspension in PBS (pH 6) readily precipitated when CPT concentration exceeded 50 μM (data not shown). In contrast, its nanoformulation (200 μM) remains suspended even after 24 h. Loading of CPT inside the vesicles was quantified by fluorescence spectroscopy. After removing the unencapsulated and loosely bound drug by centrifugation, the NVE pellet was digested with ethanol. The extract was excited at 360 nm and intensity of emitted light was recorded at 440 nm. Results showed that CPT was loaded inside the vesicles in a concentration-dependent manner. The highest encapsulation efficiency of 85 ± 9.3% was obtained when the initial drug concentration was 200 μM (Figure 4a). High encapsulation efficiency of CPT in NVEs is due to its 22145


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Figure 5. (a) Uptake of CMNVE by lung carcinoma cells A549 observed under a fluorescence microscope. Nuclei were counterstained with DAPI and images acquired at 20× magnification. Blue = DAPI and red = CM-DiI; (b) flow cytometry analysis of CMNVE-treated cells; (c) percent cell viability of free CPT and NVE-loaded CPT (NVECPT) on lung carcinoma A549 cells determined through MTT assay. Blank represents the untreated cells, and error bars are the standard error bars. (d) Apoptosis assay of free CPT and NVECPT with lung carcinoma cells A549 cells. Quadrant (I) represents relative cell population which are either dead or late apoptosis. Quadrant (III) signifies healthy population, while quadrant (IV) signifies cell population in early apoptosis.

fluorescence signal due to NVEs adsorbed onto cell surface, the treated cells were washed briefly with 0.1% Tween. As seen under a fluorescence microscope, (Figure 5a) no red fluorescence was observed in untreated cells (BLANK). In contrast, strong fluorescence was observed in cells treated with vesicles (CMNVE), and its localization around nuclei (stained blue with 4′,6-diamidino-2 phenylindole (DAPI)) strongly suggests efficient uptake of NVEs by cells. Uptake was further quantified by flow cytometry (Figure 5b). There was a large shift observed in red fluorescence for CMNVE compared to the blank (untreated cells). From histogram, it was determined that 93.5% of cell population showed uptake of NVEs. Thus, it can be concluded that RBC membrane-derived nanoparticles can be effectively internalized by A549 cells. 2.3.4. Cytotoxicity of CPT-Loaded NVEs with Lung Carcinoma Cells. CPT is a reversible topoisomerase inhibitor, which restricts DNA synthesis.30 However, its bioavailability is poor due to its insolubility in water and off-target side effects. To increase its bioavailability, CPT can be encapsulated inside the bilayer of NVEs, which can act as a biocompatible system to prolong its release specifically at the diseased site. Cytotoxicity of free CPT and NVECPT on A549 cells were evaluated using MTT assay. We have also examined the cytotoxicity of CPT and NVECPT on two other cancer cell lines, viz., glioma (U87MG) and cervical cancer (HeLa) cell lines. According to the previous literature, CPT and its analogues are reported for the lung carcinoma cell lines. Therefore, further experiments were performed on A549 lung carcinoma cell lines. For control, untreated cells and cells treated with unloaded NVEs (NVE) were taken. Free CPT shows cytotoxicity in a concentration-dependent manner, with 50% cell viability observed with 5 μM concentration (Figure 5c). An almost similar trend of cell cytotoxicity was observed with NVE-loaded CPT. Importantly, the cell viability of NVECPT-treated cells was slightly higher than that of free CPT for all concentrations of CPT used. A similar result was also observed in glioma and HeLa cell lines, reported in the

Supporting Information (Figure S2a,b). Since free NVEs were not cytotoxic, the above observation can be rationalized based on effective drug transport across the cellular membrane by the NVEs. Further, we examined the mode of cytotoxicity of NVECPT compared to free CPT via apoptosis assay (Figure 5d). For this, untreated cells were taken as control and all samples were labeled with propidium iodide (PI) and FITC-annexin V and analyzed through flow cytometry. A contour plot was made with y axis and x axis signifying PI and FITC positive cell population, respectively. The first quadrant (I) shows cell population which are either in late apoptosis or are dead. While the third quadrant (III) shows only the viable cell population, in the fourth quadrant (IV), only cells in early apoptosis can be noted. Since apoptosis is a systematic and slower process than necrosis, ideally cell population should migrate from quadrant III to IV to I with time. At 24 h, only 22.4 and 21.2% of cells treated with free CPT and NVECPT, respectively, were present in early apoptosis. This is slightly higher than the untreated population (14%). When seen under a microscope after 24 h, only few cells were rounded up but none of them had been detached from the surface. After 48 h of incubation, most of the cells were rounded up and some of them were seen floating in the media. This is also reflected in the apoptosis analysis; 39.4 and 53.4% of free CPT and NVECPT-treated cells, respectively, were present in early apoptosis while the population of dead cells and cells in late apoptosis increased to 12.8 and 14.8%, respectively. This subtle shift in cell population in early apoptosis with time shows the mode of cytotoxicity of the drug and its nanoformulation is via apoptosis, not necrosis. 2.4. In Vivo Studies. After in vitro studies of theranostic NVE, its compatibility was evaluated in vivo in balb/c mice. Twelve male mice were taken and randomly distributed into four groups (n = 3 mice in each group). These mice were injected with free CM-DiI and CM-DiI-loaded NVE (CMNVE) in respective groups. The fluorescence of CM-DiI was 22146


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3. CONCLUSION AND FUTURE OUTLOOK In conclusion, we demonstrate here the potential of cellular membrane to entrap simultaneously a hydrophobic drug campothecin and an amphiphilic fluorophore CM-DiI. After loading, nanosized vesicles can be formulated by extrusion, which retain the basic structural characteristics of the parent cells. It is possible to detect and quantify the drug in the presence of fluorophore as their emission spectra do not overlap. Further, these vesicles are stable and possess stealth in vitro. Vesicles do not cause hemolysis and show undetectable phagocytosis even in the presence of human serum. Additionally, these vesicles are immunosilent as they show only minimal elevation of proinflammatory cytokines. Further, these vesicles show a high encapsulation efficiency of CPT and are able to release it slowly over a period of 24 h, while retaining the fluorescence intensity of CM-DiI. Hence, these carriers are well suited for theranostic applications. NVEs are also internalized by A549 cells and exhibit enhanced cytotoxicity in lung carcinoma cells. Additionally, these vesicles show prolonged circulation (more than 48 h), better than pegylated liposomes and effective tissue accumulation in various vital organs in animal model, which suggest their compatibility for in vivo applications, especially in the tumor models. We envision that this system can be further developed as personalized drug carriers, which will have superior biocompatibility and optimum drug-delivery properties comparable to synthetic vesicles. These systems can be further explored for delivery of other hydrophobic drugs with different chemical complexities. It will be of great significance if one can form stable nanovesicles from RBCs discarded by blood banks.

monitored in blood obtained from retro-orbital vein at 4, 24, and 48 h. In the case of free CM-DiI injected mice, no signal of the dye in blood was detected after 4 h. Therefore, further studies could not be continued in CM-DiI-treated mice. The pharmacokinetics and biodistribution of CMNVE in the mice are shown in Figure 6. NVEs have % ID/g values of 3.88 ±

Figure 6. Pharmacokinetics and biodistribution of CM-DiI-loaded vesicles (CMNVE) in male balb/c mice (6−8 weeks, 20−35 g). Concentration of nanovesicles is reported as a percentage of the injected CM-DiI dose per gram of tissue (% ID/g).

0.14, 8.78 ± 0.03, and 4.93 ± 0.13 in blood at 4, 24, and 48 h, respectively. It is significant to note that at 24 h post injection, a relatively higher retention of NVEs in blood (% ID/g value 8.78) was observed. This is significantly longer than that observed for pegylated liposomes (shown up to 15 h).9 The NVEs were further detected in a significant amount even after 48 h (% ID/g 4.93 ± 0.13). We were surprised by the long circulation time observed by the NVEs compared to other nanoparticles with stealth coating of PEG.44−46 Also, it is important to note that the λem value (at 575 nm instead of 595 nm for free dye) of CM-DiI in blood suggests that the vesicles were intact and the fluorescence is not from free CM-DiI. Thus, extended circulation lifetime of RBC-derived vesicles reveals their remarkable stealth nature in vivo and promises effective tumor accumulation and sustained release of drugs over longer time. In addition to this, accumulation of NVEs was also examined in other vital organs (i.e., brain, heart, lungs, liver, spleen, and kidney) at 4, 24, and 48 h post injection. At each time point, vesicles were found to be mostly distributed in liver and spleen, while no major accumulation of NVEs was observed in other organs. In liver, maximum accumulation of NVEs (5.06 ± 0.09% ID/g) was observed at 24 h; in contrast, their uptake was increased in spleen with time (9.99 ± 0.26% ID/g at 24 h and 11.53 ± 0.011 %ID/g at 48 h). This observation is consistent with the results reported earlier,47,48 where relatively higher accumulation of RBCs membranecoated gold nanocages was shown in spleen compared to liver. This can also be rationalized by the fact that the spleen contains a filtration mechanism, which removes unwanted red blood cells and particles from the blood.47,48 The prolonged blood circulation of NVEs also supports the transportation of NVEs from liver to spleen. 47,48 Taken together, the observations suggest that these RBC-derived nanovesicles are suitable for further in vivo applications.

4. EXPERIMENTAL SECTION 4.1. Materials. Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM), antibiotic−antimycotic, RPMI 1640 media, fetal bovine serum (FBS), 4′,6-diamidino-2 phenylindole (DAPI), trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), CM-DiI, and bicinchoninic acid assay kit (BCA) were obtained from Invitrogen. Apoptosis assay kit was purchased from BD Biosciences. Bovine serum albumin (BSA) was purchased from HiMedia. Uranyl acetate and lipopolysaccharide (LPS) were obtained from Sigma. Micro Float-A-Lyzer (8000 MWCO) was purchased from Spectrum Labs. A549 and THP-1 cells lines were procured from National Center for Cell Sciences (NCCS), Pune, India. Phorbol 12-myristate 13-acetate (PMA) and camptothecin were obtained from Merck. ELISA kits for detection of human TNF α and IL-6 were purchased from Elabscience and RayBiotech, respectively. The detection range for TNF α is 7.81−500 pg/mL with sensitivity 4.69 pg/mL. For IL-6, the detection range is 1.37−1000 pg/mL and the sensitivity is 3 pg/ mL. 4.2. Preparation of Nanovesicles (NVEs). Blood was obtained from blood bank according to institute ethical guidelines (Ethical no: P-011). The whole blood was centrifuged at 1000 × g and 4 °C for 5 min to remove the buffy coat. The freshly obtained pellet was washed with PBS thrice prior to hypotonic treatment. Packed red blood cells thus obtained were resuspended in ice-cold hypotonic solution (65 mOsm PBS) for 10 min and centrifuged at 18 000 × g and 4 °C for 15 min. The pellet was collected and further hemolyzed with 35 mOsm PBS in ice for 5 min. This step was necessary to completely remove the hemoglobin from RBCs. Then, it was centrifuged and the white pellet thus collected was resealed by resuspending it in PBS and incubating at 37 °C for 1 h. Resealed erythrocytes were extruded through 0.4 μm polycarbonate (PC) membranes using an Avanti Mini Extruder (Avanti Polar Lipids). The size of nanovesicles thus obtained was further reduced by extruding through 0.2 μm PC membranes. 22147


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suspension was incubated in a shaker incubator at 37 °C for 2 h. After that, the samples were centrifuged at 2000 g for 5 min and absorbance of supernatant was determined at 540 nm. Suspensions of RBCs with Triton X-100 and DPBS were taken as the positive control (PC) and negative control (NC), respectively. Hemolysis rate of the sample was evaluated through the following equation

4.3. Loading of Camptothecin (CPT) and Insertion of Fluorescent Lipid (CM-DiI) into Vesicle Membrane. First, CPT was suspended in PBS (pH 6.0) at a concentration of 500 μg/mL by sonication. To this suspension was added ethanolic solution of CMDiI such that the final concentration of dye was approximately 12.5 μg/mL. This cocktail was immediately added to resealed erythrocyte pellet at a volume ratio of 2:1. The resulting suspension was vortexed and incubated at 37 °C for 30 min and further incubated in ice for another 5 min. During this process, both types of hydrophobic molecules were partitioned inside the membrane, albeit some partitioning inside the aqueous core cannot be ruled out. Then, the solution was centrifuged to remove the supernatant and washed three times with PBS (pH 6.0). The resulting pellet was resuspended in an appropriate volume of PBS (pH 6.0) and extruded through PC membrane to form vesicles. During extrusion, larger “soft” erythrocytes were squeezed through nanometer-sized pores of the membrane to yield smaller particles. 4.4. Characterization of Nanovesicles. 4.4.1. Size and ζPotential. The morphology and size of vesicles were examined using transmission electron microscopy (TEM). A drop (approximately 3 μL) of vesicles suspension was added on carbon-coated 200 mesh copper grids. The sample was allowed to stand on the grid for 5 min at room temperature. After that, the extra drop of the sample was wiped and 3 μL of uranyl acetate (1% w/v) was added on the grid. After 5 min of staining, excess stain was removed and carbon grid was visualized under a transmission electron microscope (JEOL JEM1400). The size of the vesicles was also analyzed using dynamic light scattering (DLS; Malvern Nano ZS). Further, the surface charge of the particles was determined using Zetasizer. All of the measurements were done in triplicate, and standard deviation was plotted as error bars. 4.4.2. Stability Profile of NVEs. Stability of NVEs was determined in both PBS (pH 7.4) and 10% FBS (in PBS) using DLS. Samples were prepared analogously as mentioned above. NVEs were suspended in the above media and incubated at 37 °C. Size of the particles was measured at two different time points (time 0 and after 24 h incubation) using DLS and graph was plotted against time. 4.4.3. SDS-PAGE. For evaluating the membrane protein composition of NVEs, samples were ultracentrifuged at 100 000 × g for 1 h at 4 °C using ultracentrifuge (Beckman Coulter) to pellet out the vesicles and remove any protein, which could have separated from the membrane during processing. The pellet was collected and membrane proteins were denatured in 5× loading buffer by heating at 90 °C. Then, membrane proteins were separated by running the samples on 12% gel at 90 V for 3 h. Protein bands were stained with Coomassie Blue and observed under gel documentation system (UV-P). 4.4.4. Detection of Surface Marker CD47. Freshly prepared NVEs were incubated with 1% BSA in tris buffered saline (TBS) at 37 °C for 1 h to avoid any nonspecific binding of antibodies on nanoparticles surface. Then, NVEs were incubated with primary rabbit antihuman CD47 antibody at 37 °C for 1 h. After that, unbound antibody was removed by centrifugation and NVEs were washed with tris buffered saline (TBS) thrice followed by incubation with Alexa Flour 568labeled secondary mouse antirabbit antibody for 1 h. After washing with TBS, the samples were analyzed for detection of fluorescent NPs by particle size analyzer. 4.4.5. Fluorescence Properties of NVEs. CM-DiI-labeled NVEs were visualized directly under a fluorescence microscope (Olympus IX73) and the corresponding spectra were recorded by a multiplate reader (BioTek Synergy H1). About 5 μL of labeled NVEs suspension was mounted on a clean glass slide, covered with a coverslip and examined under TRITC filter in a fluorescence microscope at 40× magnification. The spectra of labeled vesicles in PBS were recorded at an excitation wavelength of 520 nm, and for positive control, spectra of free CM-DiI in ethanol were also recorded. 4.5. In Vitro Biocompatibility Studies. 4.5.1. Hemolysis. The hemolytic properties of RBC membrane-derived vesicles were evaluated using the procedure described elsewhere.35 Briefly, 400 μL of different concentrations of NVEs suspension in PBS was mixed with equal volume of 10% packed RBCs in PBS. The resulting

% hemolysis = (OD540 sample − OD540NC) × 100/(OD540 PC − OD540 NC) 4.5.2. Cytocompatibility of NVEs with Lung Carcinoma A549 Cell Lines. Cytocompatibility of NVEs was evaluated by MTT assay. For this, A549 cells were seeded in a 96-well plate at a seeding density of 104 cells/well. NVEs, in different concentrations, were suspended in complete medium and added to cells. After 48 h of incubation, the medium was removed and 100 μL of MTT (0.5 mg/mL) in the medium was added to each well. When violet crystals were formed, MTT was removed and crystals were dissolved in dimethyl sulfoxide (DMSO) and absorbance was recorded at 550 nm. Percent cell viability was calculated using the given formula and a bar graph between percent cell viability and NVEs concentration was plotted. Untreated cells were taken as the control, and all experiments were performed in triplicate. 4.5.3. Protein Adsorption from BSA Solution. The vesicles were prepared and incubated with 1 mg/mL BSA solution in PBS at 37 °C for 1, 6, and 12 h. For control, equal volumes of RBCs and ghosts were taken. At each time, the samples were centrifuged and concentration of BSA in the supernatant was analyzed spectrophotometrically through BCA assay. Percentage of BSA adsorbed was calculated using the following formula % BSA adsorption = (OD562 supernatent/OD562 stock) × 100 4.5.4. Opsonization and Phagocytosis. For this, a procedure similar to that reported earlier was adopted.41,49 THP-1 cells were grown in RPMI 1640 medium supplemented with 10% FBS and antibiotics at 37 °C in a 5% CO2 incubator. For differentiation into macrophages, THP-1 cells were incubated with 100 nM PMA. After 24 h, the medium was removed and cells were subsequently incubated with a fresh medium for 48 h. The fluorescently labeled nanovesicles (CMNVE) were preincubated with human serum and incubated with macrophages for 1 and 4 h, respectively. For control, untreated cells and cells incubated with CMNVE without pretreatment with serum were taken. Then, the medium was removed and the cells were washed with PBS. Subsequently, the cells were fixed with 4% PFA, washed, scrapped, and then analyzed through flow cytometry.41 4.5.5. Cytokine Release Assay. 4.5.5.1. ELISA for TNF α and IL-6. THP-1 cells were cultured in 24-well plates at a seeding density of 1.2 × 105 cells and differentiated using the above procedure. Meanwhile, nanovesicles were formulated in sterile conditions and differentiated cells were incubated with 600 μL of media containing two different concentrations of nanovesicles. Lipopolysaccharide (LPS) at a concentration of 1 μg/mL was taken as the positive control. Cell supernatant was harvested at 12, 24, and 48 h of incubation, and the concentration of cytokines was determined using the ELISA technique. Concentrations of cytokines were calculated with respect to a standard calibration curve plotted with reference recombinant protein. The measurement was done in duplicate, and the concentration of cytokine was expressed in pg/mL. 4.6. Theranostic Properties of NVEs. 4.6.1. Loading Efficiency of CPT. The estimation of CPT loaded inside the vesicles was made through fluorescence.29 For this, a calibration curve of CPT in ethanol was plotted at the excitation maximum of 360 nm and emission wavelength of 440 nm. The curve was fitted into a straight line by linear regression method. Resealed erythrocytes were incubated with varied concentration of CPT suspension (in PBS pH 6.0) at fixed erythrocytes-to-drug volume ratio of 1:2. Then, it was centrifuged to remove the unencapsulated drug and washed three times with PBS (pH 6.0), followed by extrusion through PC membrane. The resulting nanoformulation was digested in ethanol and the amount of loaded 22148


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ACS Applied Materials & Interfaces drug was estimated through fluorescence. Loading efficiency was calculated by the formula

PI. Then, the cells were analyzed through FACS for relative population of cells in early apoptosis and late apoptosis or dead cells. 4.7. In Vivo Studies. Twelve male balb/c mice (6−8 weeks, 20− 35 g) were obtained from Central Animal Facility, All India Institute of Medical Sciences (AIIMS), India. The animals were randomly divided into four groups (n = 3 per group). All animal experiments were carried out in compliance with the guidelines of CPCSEA (committee for the purpose of control and supervision of experiments on the animals), Govt. of India, and all of the study protocols were approved by the Institutional Animal Ethics Committee (124/IAEC1/2019). Free CM-DiI and CM-DiI-loaded NVEs (CMNVE, 100 μL) in PBS were injected through the tail vein in each mouse of the respective group. At varying time point after injection (i.e., 4, 24, and 48 h), the mice were sacrificed to collect the blood and harvest other vital organs (brain, heart, lungs, liver, spleen, and kidney). Then, plasma was separated out from the blood by centrifugation and fluorescence of CM-DiI was measured at Ex/Em 530/575 nm. All vital organs were homogenized in PBS (0.1 g of tissue in 1 mL of PBS), centrifuged, and NVEs were quantified in supernatants. The CM-DiI content in each organ was expressed as a percentage of the injected CM-DiI dose per gram of tissue (% ID/g).

loading efficiency = (amount of drug loaded /amount of drug taken) × 100 4.6.2. In Vitro Release Profile of CPT and CM-DiI from CMNVECPT. The release of CPT and CM-DiI from nanovesicles was determined by dialyzing the co-loaded NVEs through Micro Float-A-Lyzer (8000 MWCO) placed in PBS (pH 7.4). Briefly, Float-A-Lyzer containing 600 μL of sample was placed in 400 mL of PBS as wash buffer. At each time point, 10 μL of sample was taken out from the bag to know the concentration of the remaining drug and dye using fluorescence. Percent release of CPT was determined using the formula % release = 100 − {(fluorescence of CPT at timet /fluorescence of CPT at time zero) × 100} While retention of CM-DiI inside the vesicles was determined as normalized fluorescence according to the formula

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normalized fluorescence = (fluorescence of CM‐DiI at timet

ASSOCIATED CONTENT

S Supporting Information *

/fluorescence of CPT at time zero) × 100

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b04827. Characterization of NVEs, phagocytosis, release profile of free drug, and dye and cytotoxicity assay on U87MG and HeLa cells (PDF)

4.6.3. Cell Uptake of CMNVE by Lung Carcinoma Cells. Uptake of CM NVE in lung carcinoma cell line A549 was evaluated qualitatively and quantitatively by fluorescence microscopy and flow cytometry, respectively. For microscopy, 6 × 104 cells were seeded in a 24-well plate and cultured overnight in complete medium. Then, the medium was replenished with 500 μL of media containing CM-DiI-labeled vesicles. After 3 h incubation, the medium was removed, and the cells were washed with PBS three times and then treated with 300 μL of 0.1% Tween 20 in PBS for 30 s to remove any surface-bound vesicles. Further, the surfactant was removed and the cells were washed with PBS three times. Then, the cells were fixed with 300 μL of 4% (w/v) paraformaldehyde for 15 min, followed by three washes with PBS. For counterstaining nuclei, the cells were incubated with 300 nM DAPI for 1 min, washed three times with PBS, and observed under TRITC filter in a fluorescence microscope. For quantification of uptake by flow cytometry (FACS, BD Biosciences), a similar procedure of cell seeding and sample preparation was followed as mentioned above. After incubating the samples for 3 h, the cells were washed three times with PBS and trypsinized with 250 μL of trypsin. The cell suspension thus obtained was centrifuged at 700 rpm for 5 min, washed, and resuspended in 10% FBS in DPBS, followed by quantification using flow cytometry. For all uptake experiments, cells incubated with complete media were taken as the negative control. 4.6.4. Toxicity of NVECPT with Lung Carcinoma A549 Cell Lines. The toxicity of CPT-loaded NVEs (NVECPT) on A549 cell lines was evaluated using MTT assay. For this, the cells were seeded in a 96well plate at a seeding density of 104 cells/well and grown in complete DMEM overnight. Then, the medium was replenished with fresh media containing different concentrations of NVECPT and incubated further for 48 h. The medium was then removed and MTT reagent (0.5 mg/mL) in DMEM was added and incubated for another 2 h. Formazan crystals thus formed were solubilized in DMSO and their absorbance was recorded at 550 nm. For comparison, free CPT in equivalent concentrations was also evaluated for its cytotoxicity. All samples were taken in triplicate and percent cell viability was calculated using the formula

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Neetu Singh: 0000-0002-7880-4880 Author Contributions

S.M., S.D., and P.S. performed the experiments, S.M., S.D., and N.S. wrote the manuscript. N.S. conceived the idea and provided overall guidance and feedback. All authors reviewed the manuscript for final approval. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Prof. Veena Koul for providing DLS facilities. The authors acknowledge Department of Biotechnology (DBT), India, for the financial support through project BT/PR21902/NNT/28/1240/2017. They also acknowledge the CRF, IIT Delhi, for various characterizations, and Central Animal Facility, AIIMS, New Delhi, for providing animal facilities to carry out in vivo works. S.M. acknowledges UGC for fellowship support. The authors thank Priyanka Nair and Debajit Dey from Kusuma School of Biological Sciences (KSBS), IIT Delhi, for providing them ultracentrifuge facilities and their help in establishing the initial experimental setup. S.M. thanks Tejinder Kaur and Smita Patil for their help in writing the manuscript. They also thank Zurryat Fatima from Malvern Panalytical for providing DLS facilities.

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percent cell viability = (OD550 sample/OD550 untreated cells) × 100

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For understanding the mode of toxicity (apoptosis or necrosis), cells treated with free and CPT-loaded nanoparticles were analyzed through apoptosis assay. Briefly, cells treated with 1 μM CPT (free and NVECPT) for 24 and 48 h were stained with FITC-annexin V and 22149


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