International Journal of Pharmaceutics 471 (2014) 349–357

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Characterization of lysosome-destabilizing DOPE/PLGA nanoparticles designed for cytoplasmic drug release Resham Chhabra a , Andreas M. Grabrucker a,b , Patrizia Veratti c , Daniela Belletti c , Tobias M. Boeckers b , Maria Angela Vandelli c , Flavio Forni c, Giovanni Tosi c, Barbara Ruozi c, * a b c

WG Molecular Analysis of Synaptopathies, Neurology Department, Neurocenter of Ulm University, Ulm, Germany Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Pharmaceutical Technology, Te.Far.T.I. group, Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 183, Modena, Italy

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 April 2014 Accepted 28 May 2014 Available online 2 June 2014

Polymeric nanoparticles (NPs) offer a promising approach for therapeutic intracellular delivery of proteins, conventionally hampered by short half-lives, instability and immunogenicity. Remarkably, NPs uptake occurs via endocytic internalization leading to NPs content's release within lysosomes. To overcome lysosomal degradation and achieve NPs and/or loaded proteins release into cytosol, we propose the formulation of hybrid NPs by adding 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as pH sensitive component in the formulation of poly-lactide-co-glycolide (PLGA) NPs. Hybrid NPs, featured by different DOPE/PLGA ratios, were characterized in terms of structure, stability and lipid organization within the polymeric matrix. Experiments on NIH cells and rat primary neuronal cultures highlighted the safety profile of hybrid NPs. Moreover, after internalization, NPs are able to transiently destabilize the integrity of lysosomes in which they are taken up, speeding their escape and favoring cytoplasmatic localization. Thus, these DOPE/PLGA-NPs configure themselves as promising carriers for intracellular protein delivery. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Nanoparticles (NPs) PLGA DOPE Stability Endo-lysosomes Drug delivery

1. Introduction Proteins such as enzymes and peptide hormones are considered as potent therapeutics owing to their biological roles. However, the effective intracellular delivery of exogenous proteins is limited by their short half-lives, instability and immunogenicity (Pisal et al., 2010). At the same time, polymeric nanoparticles (NPs) are regarded as highly promising protein delivery systems due to various advantages, such as efficient targeting, protection of loaded proteins from being metabolized and assurance of a timed and quantitatively controlled release of the embedded peptide (Misra et al., 2003). Therefore, the last few decades have witnessed an unprecedented advance in the development of NPs with excellent potential for intracellular protein delivery including liposomes, polymeric or gold nanoparticles, and carbon nanotubes (Du et al., 2012).

* Corresponding author. Tel.: +39 059 2055128; fax: +39 059 2055131. E-mail address: [email protected] (B. Ruozi). http://dx.doi.org/10.1016/j.ijpharm.2014.05.054 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.

Protein therapy mediated by such nanocarriers achieved varying degrees of success depending on their mechanism of internalization. The majority of cellular uptake of NPs occurs via the endocytic pathway (Panyam et al., 2002; Sahay et al., 2010) resulting in the entrapment of the carrier and its content in the endosome and eventually in the lysosome, where active degradation processes take place. As a result, only a small fraction of unaffected substance appears in the cell cytoplasm leading to very low bioavailability (Griffiths et al., 1988; Hillaireau and Couvreur, 2009). Here, we used injectable nanoparticulate drug carriers made of poly-lactide-co-glycolide (PLGA), extensively demonstrated to be able to deliver proteins and peptides (Zhang et al., 2013; Danhier et al., 2012; Patronidou et al., 2008). Notwithstanding these findings, only few articles investigated the transition of NPs from the “endo-lysosomal compartments” to the cytosol, which is often the preferred target destination of the encapsulated active substance. In recent studies (Duncan and Richardson, 2012), the intracellular trafficking of NPs was deeply characterized in order to define a pathway for cell entrance and for the subsequent fate within endo-lysosomal vesicles. As a large amount of NPs are

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submitted to dynamic transport from early endosomes to lysosome, thus not being able to escape from the endo-lysosomes deactivating pathway, here we try to find new strategies for lysosomal escape. Possible strategies to overcome lysosomal degradation and achieve the release of entrapped cargo into the cytosol could be based on pH buffering effect, flip–flop mechanism, pore formation, photochemical internalization and membrane-destabilization by use of peptides that mimic the endosomal disruptive properties of fusogenic sequences of viral fusion proteins (Liang and Lam, 2012). In this work, we decided to take advantage of pH sensitive 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by producing DOPE/PLGA-NPs. DOPE is a helper lipid which promotes the flip–flop mechanism, commonly used to initiate the fusion of the lipoplexes with the endosomal membrane. This way, the release of DNA and/or lipoplex into the cytoplasm during gene transfection is favored. The role of DOPE in destabilizing the endosome, enhancing the transfection efficiency, was attributed to its ability to undergo transition from bilayer to inverted hexagonal structures at low pH, which are known to catalyze the fusion process with the endosomal membrane (Elouahabi and Ruysschaert, 2005). pH sensitive liposomes have already been shown to effectively deliver fluorescent probes (Straubinger et al., 1985) and several active substances (Ishida et al., 2006; Navarro et al., 2012) into the cytoplasm in vitro. In the present study, in order to display a drug delivery system able to achieve an efficient delivery of proteins into the cytoplasmatic compartment, we formulated and characterized NPs produced by a mixture of PLGA with different concentrations of DOPE as starting materials. Firstly, we characterized the new hybrid systems, taking into consideration the formulability and stability from a technological point of view. Then, given the therapeutic potential of NPs, we investigated whether control NPs (PLGA-NPs) or DOPE/PLGA-NPs alter lysosomes to enable NPs escape into the cytoplasm. Moreover, we investigated the safety profile of DOPE/PLGA-NPs formulations on NIH and rat primary neuronal cells and set up the adequate conditions to preserve the cellular viability. 2. Materials and methods 2.1. Materials As reactives, poly(D,L-lactide-co-glycolide) acid (PLGA-RG503), PLGA conjugated with rhodamine-B-piperazine (Rhod-PLGA) (Tosi et al., 2012), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), polyvinyl alcohol (PVA), trehalose dehydrate, Dulbecco's modified Eagle's medium (DMEM), heat-inactivated fetal bovine serum (FBS), and Dulbecco's phosphate buffered saline (D-PBS) were used. For details on these and other chemicals (i.e. antibodies

and products for immunochemistry), please see Supplementary Information – Methods. 2.2. NPs preparation: characterization, content of PVA, content of DOPE and stability NPs were obtained using a water-in-oil-in-water (w/o/w) solvent emulsion method (Belletti et al., 2012; Voltan et al., 2013) to mimic the best conditions able to encapsulate high amounts of protein/peptides. For NPs formulation and purification procedures, please see Supplementary Information – Methods. For clarity, PLGA NPs, NPs-1, NPs-2, NPs-3 and NPs-4 indicate the samples obtained using 0.5, 2.5, 5 and 20% (w:w) of DOPE per 100 mg of NPs, respectively, while Rhod PLGA-NPs, Rhod NPs-1, Rhod NPs-2 and Rhod NPs-3 refer to the rhodamine labeled samples (Table 1). All NPs batches were characterized in terms of particles size (Z-average) and zeta potential (Z-pot), surface morphology, determination of the amount of PVA residual (Joshi et al., 1979) and yield, as reported in Supplementary Information – Methods. The amount of DOPE forming DOPE/PLGA mixed NPs was evaluated after purification by a direct method. The results are expressed as the percentage content of DOPE in the system i.e. mg of DOPE incorporated in 100 mg of NPs, or as percentage of incorporation efficiency (IE%) which is obtained by considering the embedded mg of DOPE compared to mg of DOPE added 100. Full description of the methods is given in Supplementary Information – Methods. NP stability was evaluated under storage and simulated physiological conditions as reported in Supplementary Information – Methods. 2.3. Cell cultures and treatments Primary rat cortical cell culture and NIH 3T3 mouse fibroblast cells were used in these experiments and maintained as described before (Grabrucker et al., 2009). For details, please see Supplementary Information – Methods. Eleven days after seeding the cortical neurons (11 DIV), culture medium was replaced with fresh medium containing unlabeled NPs (PLGA NPs) or DOPE/PLGA NPs (NPs-1, NPs-2, NPs-3) at a concentration of 625 mg/ml (primary cultures) or 250 mg/ml (NIH cells). After 6 h, the medium containing NPs was removed and cells were incubated in fresh medium devoid of NPs for 1–6 days. NIH cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal calf serum. Cells were grown on commercially available chamber-slides (Nunc Lab-Tek, Fisher Scientific, Schwerte, Germany) or 24 well plates with glass coverslips treated with poly-L-lysine (0.1 mg/ml; Sigma). Cells were incubated in medium containing rhodamine labeled or unlabeled NPs (Rhod PLGA-NPs or PLGA-NPs and Rhod NPs-1, Rhod NPs-2, Rhod NPs-3 or NPs-1, NPs-2, NPs-3) for 1–6 days.

Table 1 Physico-chemical characterization of NPs with expression of relative theoretical content of polymers/lipids used in the starting material mixture. All the batches were produced with a total amount of polymer/lipids of 100 mg. Details regarding re-constituted freeze-dried nanoparticles of samples NPs-4 are not reported in the table due to poor recovery and the difficulty in purification. Sample name

Z-average nm (SD)

PDI (SD)

PLGA-NPs Rhod PLGA- NPs NPs-1 Rhod NPs-1 NPs-2 Rhod NPs-2 NPs-3 Rhod NPs-3 NPs-4

205 198 215 207 226 198 217 191 197

0.08 0.12 0.11 0.09 0.12 0.11 0.11 0.09 0.26

(12) (16) (18) (10) (21) (17) (9) (16) (39)

(0.01) (0.01) (0.02) (0.02) (0.02) (0.03) (0.01) (0.02) (0.07)

Z-pot mv (SD) 12 17 15 14 11 12 8 9 13

(2) (2) (2) (1) (2) (2) (3) (2) (5)

Re-constituted freeze-dried nanoparticles

Z-average nm (SD)

PDI (SD)

Z-pot mV (SD)

233 (18) 225 (10) 257 (24) 261 (32) 269 (31) 240 (15) 273 (31) 242 (24) N.C.

0.12 (0.02) 0.16 (0.02) 0.12 (0.02) 0.13 (0.01) 0.18 (0.02) 0.18 (0.01) 0.23 (0.02) 0.15 (0.11) N.C.

10 (5) 13 (4) 13 (3) 10 (1) 10 (2) 14 (4) 11 (4) 12 (1) N.C.

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2.4. Immunocytochemistry and determination of cell death (apoptosis versus necrosis) For immunofluorescence, the NIH cells were fixed with 4% paraformaldehyde (PFA)/1.5% sucrose/PBS at 4  C for 15 min and processed for immunocytochemistry, as reported in Supplementary Information – Methods. In order to determinate the cell death (apoptosis versus necrosis), primary cortical neurons from rat cortex were seeded on a 24 well plate and apoptosis and necrosis were analyzed using apoptotic/necrotic/healthy cells detection kit (Promokine) according to the manufacturers guidelines. For full details, please refer to the Supplementary Information – Methods. 2.5. Protein biochemistry and measurements and statistical analysis To obtain homogenate from treated cortical cultures, cells were lysed and homogenized in lysis buffer (150 mM NaCl, 1% Triton1 X100, 50 mM Tris–HCl, pH 8.0) containing protease inhibitor (Roche, Mannheim, Germany). Cell debris and nuclei were removed by centrifugation at 3200 rpm for 10 min resulting in supernatant S1 (soluble fraction) and pellet P1 (membrane associated fraction). Protein concentration of S1 was determined by Bradford protein assay. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes (GE Healthcare, Freiburg, Germany). Immunoreactivity was visualized using HRP-conjugated secondary antibodies and the SuperSignal detection system (Pierce, Upland, USA). For Measurements and statistical analysis, please refer to Supplementary Information – Methods. 3. Results 3.1. Chemico-physical characterization of different nanoparticles formulations 3.1.1. Test of DOPE/PLGA ratio: technological optimization of purification and formulation parameters Aiming to optimize the NP formulation, the removal of the excess of both PVA and the un-formulated lipid is a first need. Thus, the most useful purification procedure was set up in order to correctly study both the stability and the structure of hybrid nanoparticles. In this view, we purified DOPE/PLGA NPs both by gel chromatography and centrifugation methods. Regarding PVA residual, we noted that regardless of the purification process adopted, the percentage of PVA residual stably associated with NP structures, ranged between 4 and 6% (data not shown). Thus, no differences were ascribed to the different purification processes in terms of efficacy in PVA removal. On the contrary, regarding the purification of NPs from the free or un-formulated DOPE, the centrifugation seemed to be more effective than gel-filtration chromatography, which was not able to remove the lipid adsorbed on the surface of the NPs. The quantification of DOPE remaining entrapped into NPs purified by using centrifugation process, demonstrated a correlation between the amount of DOPE added to PLGA during formulation and the % of DOPE stably incorporated into our systems (Fig. 1A, gray bars). In fact, the percentage of DOPE was found to be 100% (w/w) in NPs-1 (corresponding to 0.5 mg/100 mg of NPs), 71% (w/w) in NPs-2 (corresponding to 1.85 mg/100 mg of NPs) and 56% (w/w) in NPs-3 (corresponding to 2.8 mg/100 mg of NPs) of the total lipid used in NPs formulation. In NPs-4, DOPE was added in wide excess, therefore it arranged into autonomous lipidic structures. As demonstrated in literature, lipidic structures such as liposomes or micelles show a smooth surface and a collapsed structure particularly when composed of lipid having the

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lowest phase transition temperature (e.g. DOPE) that gives high fluidity of membrane (Ruozi et al., 2009, 2011). The AFM images and particularly the “Error Signal” image demonstrate that this sample was heterogeneously characterized by the presence of lipid structures and spheres with wrinkled surface, probably polymeric particles, (Fig. 1C, in e and e1) and then poorly reproducible. This excess of DOPE was cleavable from the NPs only exploiting their different retention times by applying gel-chromatography. After the removal of the excess of DOPE (free or organized in micelles or liposomes) from this sample, the amount of DOPE stably formulated with PLGA was close to 2.9 mg/100 mg of NPs (Fig. 1B, gray bars). These data seem to demonstrate that, independently from the DOPE amount in formulation, no more than 3% of lipid (DOPE) can be stably formulated into PLGA NPs. Unfortunately, the percentage of NPs-4 recovery (yields%) after gel chromatography (Fig. 1B, point-line) was unsatisfactory if compared to those of other samples purified by centrifugation (Fig. 1A, point-line). Thus, considering the poor recovery and the difficulty in purification, the sample NPs-4 was eliminated from subsequent technological and in vitro tests. As a consequence, we decided to characterize and test only the samples collected after purification by centrifugation. PLGA-NPs and hybrid NPs-1, NPs-2 and NPs-3, purified and re-suspended in water, were initially characterized in terms of size, surface charge and morphology and subsequently their stability during storage and in serum conditions were tested. 3.2. Physico-chemical characterization of NPs for "in vitro" experiments The data on chemico-physical characterization of NPs batches (Table 1) evidenced that all the formulations (unlabeled and labeled) were well-formed with average diameters close to 200 nm and low polydispersity index values (PDI) suggesting a homogeneous size distribution. Hybrid NPs (NPs-1, NPs-2, NPs-3) preserved the negative Z-pot, typical of PLGA-NPs in which the surface was partially masked by the residual PVA. However, the Z-pot of NPs-3 became less negative in respect to the PLGA-NPs as well as Rhod NPs-3 in respect to the Rhod PLGA-NPs. This event could be explained considering the close cohesion between the chains of PLGA and the lipidic DOPE well assembled into the matricial structure. In contrast to PLGA-NPs, hybrid systems showed a complicated morphology and a rough surface, supporting the hypothesis of the lipid integration in the polymeric matrix. In fact, as described by “Error Signal” images (Fig. 1C, lower panel), when the percentage of DOPE in the matrix systems exceeded 1.5% (NPs-2 and NPs-3 in c1 and d1, respectively), NPs were much more flattened with no homogeneous surface and fragmented frames. 3.3. Stability of nanoparticles – storage stability All the NP suspensions, purified by centrifugation, appeared stable for at least 7 days when stored at 4  C, as confirmed by PCS analysis (Fig. 1D). In fact, no substantial changes in Z-average and a narrow size distribution (Di90) were observed during the first week after NP preparation. Amongst the samples analyzed, PLGANPs were much more stable then hybrid systems as confirmed by a significant particle agglomeration especially of NPs-2 and NPs-3 samples, notwithstanding the electrostatic repulsion between NPs was preserved by the increase of negative Z-pot. Size distribution and AFM images nicely describe this instability. In particular, beside a remarkable fluctuation of (Di90) values, the morphological AFM images reported in Fig. 1 demonstrate that, after 30 days, hybrid NPs were more flattened and aggregated compared to the “time 0” samples, while PLGA-NPs remained homogeneously

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Fig. 1. Characterization of novel DOPE/PLGA-NPs. (A, B) Content of DOPE (bars) and yield% (line) of nanoparticles purified by centrifugation (A) and gel chromatography (B). (C) Topographical (upper panels) and related “Error Signal” AFM images (lower panels) of (a) PLGA-NPs, (b) NPs-1, (c) NPs-2, (d) NPs-3, (e) NPs-4. **indicate liposomes/ micelles, *indicates hybrid nanoparticles. (D) Stability of NPs (PLGA-NPs, NPs-1, NPs-2, and NPs-3) in suspension during storage at 4  C; Z-average (left), Di90 (middle) and Zpot (right panel) after 1–30 days. The content of DOPE (mg of DOPE/100 mg of NPs) is significantly reduced after 30 days in NPs-2 and NPs-3. Right: AFM images of PLGA-NPs, NPs-2 and NPs-3 after 30 days. (E) Stability of NPs (PLGA-NPs, NPs-1, NPs-2, and NPs-3) in serum. Z-average (upper panels) and PDI (lower panels) of NP suspensions (left), and lyophilized and re-suspended NPs (right) in serum.

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dispersed on mica surface. The vesicles were plunged into an unformed material, probably DOPE, just released from the hybrid NPs. Moreover, another confirmation of the structural change in hybrid samples over time was given by the variation of the content of DOPE. In NPs-1 (0.4 mg/100 mg of NPs), in which the amount of DOPE formulated was very low (0.5 mg/100 mg of formulation), DOPE and PLGA seemed to remain steadily formulated into NPs-1 during storage. On the contrary, a progressive release of DOPE from the polymeric matrix, with a consequent loss in DOPE content, was observed during the storage of NPs-2 (the content of DOPE decreased from 1.8  0.3 mg to 1.3  0.05 mg of DOPE/100 mg of formulation) and NPs-3 (from 3.0  0.4 mg to 2.3  0.1 mg/100 mg of formulation). All the samples were lyophilized to improve their long term stability in presence of trehalose (1:1 w:w trehalose/NPs) and were reconstructed in water and analyzed at time 0 (see Table 1). The Z-average of freeze-dried DOPE/PLGA-NPs increased approximately 20% compared to a corresponding NPs suspension. Only a slight increase in the polydispersivity index was observed, confirming the maintained homogeneity of freeze-dried samples. Lyophilized samples stored at 20  C for 180 days were easily re-suspended and the initial properties of NPs were preserved. This storage temperature did not influence particle size when nanoparticles were lyophilized with trehalose. In fact, Z-average diameter of the freeze-dried NPs was less than 300 nm and the PDI value ranged between 0.2 and 0.35 for all formulations and for any time points. No DOPE loss during storage was observed (data not shown). 3.4. Incubation stability The incubation of sample (NPs suspensions) for 2 h at 37  C in culture medium supplemented with serum (DMEM + FBS + PS) did not significantly change the Z-average and the PDI values of both PLGA NPs and all hybrid systems (Fig 1E, panel 1 and 2), indicating a maintenance of the homogeneous dispersion features of the samples in a simulated biological environment. The moderate fluctuation of the particle size (Fig 1E, panel 1) and particularly of the PDI values (Fig 1E, panel 2) owing tothe incubation time might be explained by the association/dissociation processes of serum proteins onto the NP surface. The fluctuation at this serum concentration (10%) and for this incubation time (120 min) seemed negligible. Moreover, when re-suspended freeze-dried NPs were tested for stability in serum, only minor changes in average diameter and PDI values (Fig. 1E panel 3 and 4, respectively) were observed, confirming the preserved stability of hybrid systems in serum. 3.5. Determination of an effective non-toxic minimum PLGA/DOPENPs concentration In a first set of experiments, we wanted to determine the minimum concentrations of DOPE/PLGA that induce changes in lysosome – associated proteins in cortical primary neurons. The use of neurons prevents the influence of cell divisions on the amount of intracellular NPs. The same number of neurons was plated for all the experiments. We quantified the levels of lysosome-associated membrane protein 2 (LAMP2). LAMP2 is mainly localized to the lysosome membrane and has been suggested as a marker for lysosomal storage disease (Hua et al., 1998) in the past. Thus, alterations in the levels of LAMP2 are indicative for lysosomal alterations within cells. In parallel, we evaluated the levels of cleaved casapse-3, cleaved PARP and cytoplasmic cytochrome c to evaluate the toxicity of the respective DOPE/PLGA concentrations regarding their potential to induce cellular apoptosis. The activation of caspase (cysteine–aspartic acid protease) 3 (CASP3) plays a central role in the mechanisms of apoptosis. Thereby, caspase-3 can be activated both by extrinsic

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(death ligand) and intrinsic (mitochondrial) pathways (Salvesen, 2002) and is either partially or totally responsible for the proteolytic cleavage of many key proteins involved in cellular apoptosis, such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP, see below). Activation of caspase-3 requires proteolytic processing (cleavage) of its inactive form into activated p17 and p12 fragments. Thus, the amount of cleaved caspase-3 provides a read-out for apoptotic processes initiated within a cell. Similarly, the amount of cleaved PARP is indicative of programmed cell death. Normally, PARP is activated in response to metabolic, chemical, or radiation-induced DNA damage. However, in case of severe damage, PARP can be cleaved by enzymes such as caspases or cathepsins that inactivate the protein. In this case, PARP promotes apoptosis (Virág et al., 2013). The cytochrome complex (cytochrome c) is a membrane – associated component of the electron transport chain in mitochondria. Cytochrome c is also involved in the initiation of apoptosis (Kilbride and Prehn, 2013). Upon release of cytochrome c to the cytoplasm, the protein binds apoptotic protease activating factor-1 (Apaf-1), thereby inducing mechanisms finally leading to cell death. Thus, the amount of cytoplasmic cytochrome can also be used as a read-out for apoptotic processes initiated within a cell. Three formulations of NPs containing different amounts of DOPE (0.5%, 1.85% and 2.8% named NPs-1, NPs-2 and NPs-3, respectively) were compared to PLGA-NPs (Fig. 2). Moreover, NPs treated cells were compared to untreated cells (Fig. S1A). Our results show that while NPs-1 did not significantly alter LAMP2 levels compared to PLGA-NPs, the application of NPs-2 lead to a slight decrease after 4 days of treatment (Fig 2A, lower right panels). The best results were obtained using NPs-3. Here, we detected a significant decrease of LAMP2 levels at day 4 compared to levels at day 1. Moreover, LAMP2 concentrations increase after day 4 until day 6 with significant difference to day 4 but not day 1 of treatment (Fig. 2A). No such decrease was observed using NPs without DOPE. The analysis, whether PLGA-NPs or DOPE/PLGA-NPs are toxic for cells, or whether the possible decrease of lysosomes induces programmed cell death, shows no differences in the levels of cleaved caspase-3, cleaved PARP, and cytochrome c between PLGA-NPs and NPs-1 or NPs-2 (Fig 2B–D, lower right panels). Similarly, no significant difference between PLGA-NPs and NPs-3 could be detected in terms of levels of cleaved PARP and cytochrome C (Fig. 2C,D). The amount of cleaved caspase-3 was significantly less after 1 day of treatment with NPs-3 compared to PLGA-NPs, which however equaled after 2 days of treatment (Fig. 2B). In general, the levels of LAMP2 were not significantly different between cells treated with NPs or untreated cells with the exception of the reduction after 4 days of treatment with NPs-3, and the levels of cleaved Caspase-3, cleaved PARP and cytochrome c were not significantly higher in cells treated with NPs compared to untreated cells (Fig. S1A–D). Given that PLGA-NPs and DOPE/PLGA-NPs are taken up into cells in a similar amount (Fig. S1E) with the exception of NPs-1, the observed differences cannot be explained by a different amount of NP load of cells. 3.6. Toxicity profile of NPs-3 The results obtained above indicate that no major apoptotic processed are active upon application of PLGA/DOPE-NPs. To further exclude that PLGA/DOPE-NPs induce cell death, we performed a necrosis versus apoptosis assay. Neuronal cells were treated for 1–6 days with PLGA-NPs and NPs-3 and the fraction of apoptotic and necrotic cells were assessed and compared to untreated control cells (Fig. 3A,B). The results show that although we detected an increased rate of necrosis directly after application in cultures treated with NPs-3, the rate of apoptosis was not

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Fig. 2. Determination of minimum DOPE/PLGA concentration that induce changes in lysosomal LAMP2 protein levels and NP toxicity profiles. (A–D) Cortical neurons were treated with PLGA-NPs or DOPE/PLGA-NPs containing three DOPE concentrations (NPs-1, NPs-2, NPs-3) for 6 h and protein lysate was prepared after day 1–6. (A) Analysis of LAMP2 levels by western blot shows NPs-1 did not significantly alter LAMP2 levels compared to PLGA-NPs, NPs-2 lead to a slight decrease after 4 days (upper right panels). NPs-3 lead to a significant decrease of LAMP2 levels at day 4 compared to day 1. LAMP2 again increases after day 4 until day 6 with significant difference to day 4. (B) No significant differences in the levels of cleaved caspase-3 between PLGA-NPs and NPs-1 or NPs-2 were detected. Cleaved caspase-3 was significantly less after 1 day of treatment with NPs-3 compared to PLGA-NPs. After 2–6 days of treatment this difference was no longer significant. (C, D) No significant differences in the levels of cleaved PARP, and cytochrome C between PLGA-NPs and NPs-1, NPs-2 or NPs-3 could be detected. Three blots were analyzed per experiment and tested for significance using t-test.

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Fig. 3. Toxicity profile of NPs-3. (A, B) Neuronal cells were treated with equimolar concentrations of PLGA-NPs and NPs-3 and compared to untreated control cells regarding their potential to induce apoptosis or necrosis. (A) Apoptotic cells were identified using Annexin V labeled with FITC (open arrow), necrotic cells (grey arrow) by ethidium homodimer III and the total number of cells (full arrow) was assessed using Hoechst 33342 labeling all nuclei. (B) A total of three optic fields of view per condition and day were analyzed. Ethanol treatment was used as positive control (not shown). Although an increased rate of necrosis directly after application of NPs-3 can be detected, the rate of apoptosis was not significantly different to controls or PLGA-NPs on days 2–6. On day 6, the rate of apoptosis was significantly increased in cells treated with PLGA-NPs, but not in cells treated with NPs-3.

significantly different to controls or PLGA-NPs on days 2–6. However, on day 6, the rate of apoptosis was significantly increased in cells treated with PLGA-NPs, but not in cells treated with NPs-3 (Fig. 3B). In general, we could not detect a significantly increased rate of cell death averaging all days of treatment after application of NPs and no significant difference between PLGA-NPs and NPs-3. 3.7. NPs-3 transiently disrupt lysosomes To investigate if the fusogenic properties of DOPE can produce a rapid release of NPs from endo-lysosomal compartment, we incubated NIH cells with labeled control NPs (Rhod PLGA-NPs, left panel) or with labeled mixed DOPE/PLGA-NPs (Rhod NPs-3, right panel) for 24 h, 48 h and 72 h. Cells were then fixed and stained for markers of early endosomes. In particular, EEA1 (Fig. 4A, lower panel) and Rab5A (Fig. 4A, upper panel), and a marker for lysosomes, Lamp2 (Fig. 4B) were used. Nor control NPs neither mixed DOPE/PLGA NPs were found to be associated with EEA1 positive vesicles, but nicely co-localize with Rab5 positive vesicles (Fig. 4A). As EEA1 is a caveolae related early endosome marker and Rab5 is a clathrin related early endosome marker, this supports the hypothesis that these NPs, independently from their composition (PLGA 100% or DOPE/PLGA mixture) are internalized by clathrin mediated endocytosis. Interestingly, considering Rhod NPs-3, there is significantly less co-localization after 72 h with the lysosomal marker compared to the control NPs without DOPE (Rhod PLGA-NPs). The intensity of LAMP2 marker also decreases with time due to the disruption of lysosomes (Fig. 4B,C). The most interesting results were obtained by analyzing lysosome-related signals. As evident (Fig. 4C), a number of Rhod PLGA-NPs are strongly accumulating inside lysosomal vesicles over time, with a mean increase of 50% up to 72 h. On the contrary, after an initial accumulation inside lysosomes (at 24 h and 48 h), a significant decrease in NPs accumulation (close to 75%) was assessed after 72 h. To investigate the fate of lysosomes in more detail, we analyzed the amount of lysosomes over time after application of NPs using LysoTracker1 (Fig. 4D). Fluorescent LysoTracker accumulates in cellular compartments with low internal pH and thus can be used to study the functionality of lysosomes (Griffiths et al., 1988). The results show a significant decrease of LysoTracker positive signals in cells after 3–4 days after NPs-3 application compared to PLGA-

NPs. At day 6 after application, the number of LysoTracker positive signals seems to recover in cells treated with NPs-3 (Fig. 4D). 4. Discussion The treatment of monogenic disorders could highly benefit from either the delivery of the disrupted gene, e.g. by gene therapy (Weatherall, 1995) or by delivery of the gene product – the encoded protein into the affected target cells. The first approach is significantly hampered by the use of viral vectors to deliver genetic material into cells that may lead to the insertion of the gene into random sites of genomic DNA with the chance of causing tumor formation. The latter one is hard to achieve given that proteins are subject to degradation before they might reach their target. However, recent approaches using NPs have shown that NPs are able to protect encapsulated substances and peptides (Chitkara and Kumar, 2013). Moreover, NPs can be modified e.g. to gain the ability to cross the blood brain barrier (Tosi et al., 2013) or even target specific cell populations (Grabrucker et al., 2011). Despite these interesting qualities, NPs are commonly taken up into cells via endocytosis and are thus surrounded by a vesicular membrane (Ahn et al., 2013). Release of peptides would therefore lead to enrichment of the encapsulated peptide trapped within an endosome. Additionally, endosomes are trafficked within the cell and might fuse with lysosomes having a low pH and resulting in protein degradation. It is therefore necessary to not only protect a peptide during delivery within a NP, but also modify NPs in a way that they can escape lysosomal degradation and release their content within the cytoplasm of cells. Here, we investigated the PLGA/DOPE hybrid NPs with respect to their ability to fulfill the aforementioned desired qualities. All the NPs samples demonstrated a good reproducibility in terms of size, surface charge and homogeneicity, also demonstrated by AFM images, clearly showing the formation of well-structured hybrid NPs. This reproducibility resulted to be independent from the addition of different amounts of DOPE in the initial mixtures (varying from 0.5 to 5% of DOPE). Our results defined a precise amount of DOPE well formulable into a stable matricial PLGA nanoparticles. This “DOPE formulability threshold” was approximately of 3% weight of formulation (NPs-3). The particles generated can be considered hybrid systems with high stability but also good flexibility useful to interact with

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Fig. 4. NP endocytosis and localization with lysosomal markers. NIH cells were incubated with culture medium containing Rhod NPs-PLGA (left panel) or Rhod NPs-3 (right panel) for 24 h, 48 h and 72 h. Cells were then fixed and stained for markers of early endosomes; EEA1 (A, lower panel), Rab5A (A, upper panel), and a marker for lysosomes, LAMP2 (B). NPs were not found to be associated with EEA1 (A, lower panel) but nicely co-localize with Rab5. There is significantly less co-localization of NPs-3 after 72 h with the lysosomal marker compared to NPs-PLGA (p < 0.0001; n = 10). The intensity of LAMP2 also decreased with time due to the disruption of lysosomes. (D) NIH cells were treated with Rhod PLGA-NPs or Rhod NPs-3 for 6 days. Untreated cells served as controls. Ten cells from each condition were analyzed and the average number of LysoTracker positive signals per cell assessed. A significant decrease of LysoTracker positive signals can be seen after 3–4 days after Rhod NPs-3 application. On day 6, the number of LysoTracker positive signals seems to recover in cells treated with Rhod NPs-3. Images are shown as inverted gray-scale.

cells. The fusogenic properties conferred by DOPE seemed preserved also when the lipid was enclosed in the polymeric matrix, as demonstrated by cellular studies in which we observed an increased ability to transiently disrupt lysosomes if compared with PLGA-NPs. To assess, if this transient disruption increases the potential of NPs to induce apoptosis or cause necrosis, we performed a comparative study in cell culture. Apoptosis and necrosis are the two major processes leading to cell death. Apoptosis is an actively regulated cellular process induced by diverse factors, while necrosis results from cellular damage leading to a loss of organelle and plasma membrane integrity. Neither does the disruption of lysosomes nor the

application of DOPE/PLGA-NPs seem to produce enough cellular stress to induce apoptotic pathways in cells or cause necrosis of cells. Additionally, we evaluated characteristic indicators of apoptosis such as cleaved caspase-3, PARP and cytochrome c using protein biochemistry. The results underline our findings that DOPE/PLGA-NPs are, despite their ability to transiently disrupt lysosomes, not significantly more toxic than PLGA-NPs. Taken together, in this study, we followed the idea of using DOPE in the formulation of PLGA-NPs to reach the goal of cytoplasmic delivery of NPs. We therefore developed and evaluated different DOPE/ PLGA-NPs, and selected the most useful for a more detailed investigation. These NPs containing 3% DOPE were found to

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transiently disrupt lysosomes without affecting cell health more in comparison to PLGA-NPs. 5. Conclusion As it was clearly described that the fate of polymeric NPs designed for protein delivery is hampered by an intracellular endosome-based deactivation, with this paper, we aimed to establish a novel formulation, featured by DOPE/PLGA mixture, able to avoid endosomal uptake and therefore letting NPs and/or loaded cargo to be released within the cytoplasmatic environment. We demonstrated that hybrid DOPE/PLGA-NPs formulated with at least 3% (w/w) of DOPE versus PLGA were capable to ameliorate the ability in destabilizing the endo-lysosomes, displaying at the same time a lower toxicity if compared with PLGA NPs. Similar to PLGANPs, hybrid DOPE/PLGA-NPs formulated with at least 3% (w/w) of DOPE did not induce apoptosis or necrosis. We therefore conclude, that these NPs might be a step forward in targeted cytoplasmatic delivery of proteins or drugs possibly useful in many future treatments of various disorders. Conflict of interest The authors declare that they have no conflict of interest. Funding AMG is supported by Baustein 3.2 (L.SBN.0083) and the Alzheimer's Association (NIRG-12-235441). GT and AMG are supported by the DAAD (Vigoni program). RC is a member of the International Graduate School Molecular Medicine at Ulm University. Authors' contributions RC and AMG performed “in vitro” experiments and drafted the manuscript with GT and BR. AMG, GT, MAV, FF, BR and TMB participated in the design of the study. PV, DB, GT and BR contributed to the preparation of NPs. All the authors read and approved the final manuscript. Acknowledgement The authors gratefully acknowledge the professional technical assistance of Katharina Mangus. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2014.05.054. References Ahn, S., Seo, E., Kim, K., Lee, S.J., 2013. Controlled cellular uptake and drug efficacy of nanotherapeutics. Sci. Rep. 3, 1997 doi:http://dx.doi.org/10.1038/srep01997. Belletti, D., Tosi, G., Forni, F., Gamberini, M.C., Baraldi, C., Vandelli, M.A., et al., 2012. Chemico-physical investigation of tenofovir loaded polymeric nanoparticles. Int. J. Pharm. 436, 753–763.

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