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Peptide-based Stealth Nanoparticles for Targeted and pH-Triggered Delivery Alessandro Ranalli, Melissa Santi, Luigi Capriotti, Valerio Voliani, David Porciani, Fabio Beltram, and Giovanni Signore Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00701 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017

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Bioconjugate Chemistry

Peptide-based Stealth Nanoparticles for Targeted and pH-Triggered Delivery Alessandro Ranalli,1,‡ Melissa Santi 1,‡ Luigi Capriotti, 2 Valerio Voliani,2 David Porciani,1,2 Fabio Beltram,1 Giovanni Signore1,2* 1 2

Scuola Nomale Superiore, Laboratorio NEST, piazza san Silvestro 12, 56127 Pisa, Italy Istituto Italiano di Tecnologia, CNI@NEST, piazza san Silvestro 12, 56127 Pisa, Italy;

KEYWORDS: stealth liposomes, aptamers, doxorubicin, cancer therapy, targeted delivery ABSTRACT Stealth agents are extensively investigated as means to prolong nanostructures residence time in the bloodstream by avoiding uptake by the reticuloendothelial system. Unfortunately, commonly used agents such as poly(ethylene glycol) can adversely impact on targeting efficiency and promote immune reaction by the host organism. Therefore, there is an increasing interest in developing biocompatible, non-PEGylated organic nanostructures able to perform targeted delivery, to increase the efficacy of liposomal technology. Here, a lipopeptide is presented that can be mixed with lipids commonly used in liposomal formulations in percentages ranging from 20% to 60% w/w. The resulting vesicles are thermally and chemically stable. The peptide coating limits serum-protein adsorption even upon prolonged incubation in pure serum in physiological conditions, outperforming PEGylated liposomes. This architecture can be easily modified to allow straightforward derivatization by standard bio-orthogonal conjugation. Upon derivatization with an antitransferrin receptor aptamer, these vesicles show highly selective cellular internalization with minimal nonspecific uptake, and pH-triggered doxorubicin release.

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Introduction The systemic administration of drugs that aspecifically diffuse within the host organism to reach their target presents known, serious disadvantages1 such as poor uptake owing to solubility issues, poor targeting, and, particularly in the case of cancer therapy, severe side effects stemming from the aspecific uptake by healthy tissues. Targeted delivery of therapeutic agents addresses these limitations by conveying a molecular cargo to a specific cellular population with high efficiency and selectivity. To this end, many platforms were investigated in the last decades, including metal-2,3 and polymer-based4,5 nanoparticles, liposomes,6 dendrimers,7,8 and smaller molecular architectures such as engineered aptamers.9 Whatever the nature of the architecture employed, these devices must be able to avoid uptake by the reticuloendothelial system (RES) and maintain their recognition capability. Indeed, any substance injected in the bloodstream undergoes extensive, largely aspecific solvation by serum proteins: this is the primary mechanism for the RES to recognize and clear foreign structures circulating in the bloodstream (opsonization)10 and can completely abolish active targeting capabilities.11,12 In some cases the composition of the protein corona was exploited to enhance internalization of nanodevices.13 However, its presence can hamper the efficacy of targeting sequences so that most nanostructures are designed to limit protein adsorption with the use of antifouling (stealthing) surface modifiers. Polyethylene glycol (PEG), the most diffuse stealth polymer, was firstly reported in the early 90s14 and since then quite a number of nanodevices6 were decorated with PEG with the aim to limit opsonization and improve circulation time in the bloodstream.15 PEGylated liposomes in particular were extensively studied and some of them are currently employed in actual


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clinical applications.15 Unfortunately, the use of PEG as stealth agent does have some issues. Firstly, PEG chains can hamper the recognition capability of targeting sequences, due to the formation of a sterically crowded surface.16 A second hurdle is represented by the recognition of PEGylated structures by the RES upon multiple administrations. Indeed, living organisms react to the introduction of PEGylated systems by generating anti-PEG IgM17 that accelerates clearance of the nanostructure starting from the second administration of the drug. This phenomenon is referred to as “accelerated blood clearance” effect18,19 and can limit the effectiveness of PEGylated nanostructures when repeated administrations are required. Thus, there is increasing interest in developing PEG-free stealth structures. To this end, several approaches were proposed which employ, among others, zwitterionic polymers,20,21 nanoparticles derived from purified cellular membranes,22,23 or natural exosomes.24 Another promising alternative is the use of short amino acid chains able to mimic composition of serum and circulating proteins. Recently, Nowinski et al.25,26 presented an interesting approach based on short zwitterionic peptides that abolish serum protein adsorption, while leaving unaltered the targeting properties brought by suitable sequences. Translation of this strategy to an organic-based drug delivery system would provide access to PEG-free, stealth nanoparticles for targeted delivery. Indeed, the use of zwitterionic lipopeptides at high concentration could afford systems able to mimic behavior –and serum stability- of natural vesicles, while maintaining the synthetic accessibility typical of liposomes. Here, we introduce a biomimetic architecture based on the synergistic conjugation between liposomes, aptamers and stealth lipopeptides that allows the targeted delivery of small drugs in living cells, avoiding degradation even in pure serum. Results and discussion We firstly designed a stealth lipopeptide (L1) able to spontaneously insert within a lipid bilayer, exposing its polar zwitterionic head towards the aqueous environment. To this end,



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we assembled the alternated Glu/Lys motif described by Nowinski, adding a lipid-like motif composed by a lysine and two palmitoyl chains to allow efficient insertion within the lipid bilayer. The overall structure of L1 is reported in Scheme 1. The lipopeptide was easily prepared by solid-phase synthesis in good yield (35% isolated yield after HPLC purification). Preliminary measurements indicated that L1 spontaneously self-assembles only at high concentration (critical micellar concentration, CMC: 230.1 µM) leading to 10-100nm aggregates. This result ruled out any possibility to employ self-assembled L1 for delivery purposes. Based on this result, we hypothesized that co-formulation with lipids could favor self-assembly and stability at low concentrations, with the lipids acting as a “lipid skeleton” which supports the stealth peptide. Concerning the lipid part, our choice of lipid composition was based on the known thermal and mechanical stability of 4:1 DSPC/Cholesterol27 mixtures that can provide stable vesicles.

Scheme 1. Structure of stealth lipopeptide L1 Our optimization of vesicle composition started from the measurement of temperature stability and surface charge of vesicles with different percentages of L1. To this end, we prepared vesicles composed of a fixed percentage (20%) of cholesterol, and different L1/DSPC ratios (varying from 0/100 to 100/0). To better compare protein adsorption and thermal stability properties, we also prepared liposomes containing 10% w/w of DSPEPEG2000-OMe) (PEG-Lipo), a composition widely employed in the realization of stealth liposomes.28 In all cases, the vesicles were extruded through a 200 nm membrane after hydration in PBS of a freeze-dried mixture of lipids and peptides; this approach was preferred


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over the more common hydration of dried films owing to the reported greater efficiency in encapsulating lipopeptides within lipid shells.29 Composition and diameter (measured by DLS) of vesicles are reported in Table 1. Agreement between calculated and effective composition of the vesicles was checked in one representative case (V40) by LC-MS. We found that encapsulation efficiency of L1 in purified vesicles was greater than 90%, in agreement with what previously reported for analogous lipopeptides.29 Table 1. composition (w/w) and physical properties of the vesicles studies in the optimization process Diameter

Zeta

(nm)1,2

Potential

PDI

Degradation

PEGDSPC

Cholesterol

L1

T (oC)

2000 (mV)1

252±3

Plain -

80

20

0

0

60

20

0

20

-

0.16±0.03

50

0.08±0.02

65

0.11±0.02

>65

0.13±0.02

>65

0.16±0.01

55

17.8±0.7

Lipo3 152±3

V203

30.1±3.3 178±3

V40

3

-

40

20

0

-

40 41.2±0.6 155±2

V603

20

V803

0

20

0

-

60 33.8±3.6 160±4

20

0

-

80 28.2±0.5



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142±2

PEG70

Lipo3 1

20

10

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-

0.10±0.02

65

0 14.8±1.3

Average of three measurements; 2 Calculated from the intensity signal; 3 20% (w/w) of cholesterol is present in each sample.

We observed that addition of L1 has a dramatic effect on both thermal stability and surface charge. Both PEGylated and plain (i.e. with 0% of L1) liposomes have mildly negative surface charge (-15 to -20 mV, Tab. 1), while insertion of L1 significantly lowers the surface charge. Interestingly, we found that surface charge depends nonlinearly on the percentage of lipopeptide in the vesicle (see Tab. 1), but it is always between -30mV and -40mV. The maximum effect is obtained with a ratio L1/DSPC/Chol 40/40/20 (V40). At higher lipopeptide percentages, the modulus of the surface charge decreases steadily, although a potential of at least of -30 mV is always observed. Note that this zeta potential closely matches values often found for exosomes and other extracellular vesicles (-30 to -50 mV).30 We also found that insertion of L1 significantly increases thermal stability, as demonstrated by the higher degradation temperature compared with plain liposomes. Indeed, formulations with 40 to 60 % of L1 are thermally stable at least up to 65 oC (the upper limit used in our thermal-stability measurements), as is the case for PEG-Lipo (Tab. 1). Interestingly, temperature stability follows a trend similar to what observed for zeta potential, with best stability achieved in the case of V40 and V60. The observed trend can be rationalized on the basis of the average distance between different L1 units on the surface of the vesicle. At low percentages, L1 coverage is not sufficient to alter significantly the surface charge of the vesicle, leading to a zeta potential that is near to that shown by plain vesicles. Conversely, when two L1 units are in close proximity on the surface of the micelle the electrostatic attraction between side chains of glutamate and lysine residues present on proximal units can partially mask the effective surface charge brought by the external (C-terminal) glutamate,


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thus lowering the overall negative charge of the vesicle. When L1 percentage is further increased, such as in V80, interaction between the charged residues leads to extensive destabilization and degradation of the vesicle in short times. To evaluate the influence of L1 on serum-protein adsorption the vesicles were incubated in physiological conditions (37oC) with pure fetal bovine serum (FBS), monitoring diameter change by DLS over an extended time. Preliminary measurements performed on pure-serum samples evidenced that protein and lipid aggregates present in FBS are small (8 and 50 nm) and well resolved from the sensibly larger synthetic vesicles (200-240nm) that could be measured with high accuracy. As expected, incubation of plain liposomes with serum does lead to extensive serum-protein adsorption, with complete degradation observed within 60 minutes. Conversely, benchmark stealth PEG-Lipo shows limited protein adsorption even after 500 minutes (Figure 1) showing limited -yet not negligible (48%)- diameter increase over 8 hours.

Figure 1. Diameter increase (measured by DLS) shown by different vesicles after incubation for 500 minutes in pure serum at 37oC, with respect to starting diameter. Diameter increase is proportional to the amount of adsorbed proteins and to protein-dependent degradation. Measurement for Plain-Lipo is performed after 50 minutes, since plain liposomes are



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completely degraded within 60 minutes. Results are the average of three different experiments. Notably, the presence of L1 even at low percentages confers stealth properties to the vesicles, as shown by the limited protein adsorption of V20. In all cases, diameter increase (a clear hallmark of adsorbed proteins) is at least comparable with what shown by benchmark PEGLipo. Notably, in the case of V40, protein adsorption is negligible (10%) during the entire incubation period (500 minutes), outperforming even benchmark stealth liposomes. Interestingly, protein adsorption on V60 appears to be higher than on V40, despite the increased amount of stealth lipopeptide. Since Tab. 1 (entries 3 and 4) shows that this trend correlates with the decrease in the surface-charge modulus, it is tempting to deduce that at concentrations higher than 40% w/w the proximity of the zwitterionic lipopeptides may lead to significant electrostatic interactions between positively and negatively charged side chains. This in turn could lead to lower net surface charge and hence to weaker stealth behavior. Overall, these stability assays provided us with clear indications that vesicle formulation of V40 (i.e. Chol/DSPC/L1 20/40/40) is the best performing in terms of thermal stability and antifouling properties. This optimized formulation was thus kept as a basis in all subsequent studies. The development of a targeted architecture for selective delivery requires the insertion of natural (antibodies, ligands, or proteins) or synthetic (aptamers, peptides) biomolecules able to perform selective recognition of their molecular target. With the aim of exploiting at best the versatility of our device, we developed a derivatizable version of our system. Compared with the direct insertion of lipid-modified biomolecules during vesicle formation, the use of bio-orthogonal handles embedded in the lipid bilayer has the distinctive advantages of greater flexibility, better synthetic accessibility, and improved control on the orientation of the


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targeting molecule (i.e. targeting unit is present only on the outer surface of the vesicle). For example, established protocols exist for the derivatization of biomolecules with thiols, which can be reacted with maleimide-based handles in very mild conditions, preserving threedimensional structure and hence functionality of proteins and antibodies. To this end, we designed a derivatizable lipopeptide composed of the same sequence used for L1, but with an additional maleimide linker at the outer terminal (i.e. the N-terminal portion opposed to the lipid chains). The synthesis of such handle was carried out in two steps (Scheme 2). Firstly, we synthesized by standard SPPS approach a fully protected version of L1 and this was subsequently cleaved from the resin in mildly acidic conditions leaving protecting groups unaltered. Next, an aminoethylmaleimide linker was conjugated by standard DIC chemistry and the final product was deprotected and purified by HPLC. The whole synthetic strategy yielded the lipopeptide-based handle (L1-Mal).

Scheme 2. synthesis of functionalizable lipopeptide L1-Mal. Asterisks indicate the presence of Boc or t-Bu protecting groups.



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This functionalized structure was used in the preparation of derivatizable vesicle (V40-Mal). To avoid adverse effects on the final surface charge of the stealth lipopeptide (see Tab. 1), the overall percentage of stealth peptide in the synthesis of V40-Mal was maintained unaltered with respect to V40. Thus, the percentage of L1 was lowered of the same amount of the added L1-Mal, maintaining a constant lipid fraction. In agreement with the vast literature on targeted liposomes, we decided to keep the w/w percentage of L1-Mal (and hence of targeting sequences) at 10%, a level sufficiently low to allow efficient targeting without altering significantly the overall properties of the vesicle. Additionally, we inserted a fluorescent lipid marker (DOPE-Rhodamine) at low concentration (1%) to provide a flag for optical readout in subsequent internalization and cell binding studies. Fluorescently labeled untargeted vesicles V40 were also prepared for comparison purposes. As expected, the presence of L1-Mal -whose negative surface charge is partially masked by maleimido groups- lowers the surface charge of the vesicle; this in turn results in the decrease of zeta potential from -35mV to -15mV (Figure 2). Interestingly, the hydrodynamic diameter of derivatized V40-Mal and parent vesicles V40 were not statistically different, indicating that the presence of up to 10% of L1-Mal does not interfere with the assembly process or with the stability of these structures (Figure 2). We decorated the exterior of the assembled vesicle with a thiol derivatized version of an anti TfR DNA aptamer (DW4),31 using standard maleimide-thiol coupling. DW4 is a rationally designed evolution of the previously reported GS24 aptamer32 that recognizes human and mouse transferrin receptor (TfR), promoting internalization in physiological conditions. TfR is a widely studied target in cancer research owing to its overexpression on the cell surface of most solid tumors and its constitutive tendency to internalize in cells by active endocytosis processes. Conjugation of the thiol-modified aptamer to the maleimide-derivatized vesicle proceeds smoothly and the purified product V40-DW4 shows, as expected, a more negative


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surface charge (-57 mV) compared with its partially neutralized precursor V40-Mal (-15 mV). The absence of statistically significant variations in vesicle diameter indicates that the supramolecular structure is maintained during labeling and purification procedures (Figure 2). Importantly, this conjugation strategy enabled us to insert multiple targeting aptamers on the surface of the nanovesicles, thus increasing the avidity and the affinity of DW4 towards TfR.33,34

Figure 2. Physico-chemical properties of functionalizable, functionalized, and benchmark vesicles. The targeting efficiency of V40-DW4 was tested on MiA PaCa-2 cells, a human tumor pancreatic cell line which is known to overexpress transferrin receptor.9 We evaluated the internalization extent by flow cytometry with the aim of obtaining quantitative results on the internalization efficiency. Not surprisingly, we observed that aspecific uptake of untargeted



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V40 is negligible, while the presence of aptamer targeting units on the surface promotes extensive internalization (median shift of fluorescence signal compared with the control: 8% and 23% for V40 and V40-DW4, respectively, Figure 3a). Furthermore, vesicle intracellular distribution was observed by means of confocal fluorescence microscopy, evidencing that part of the low fluorescent signal arising from untargeted vesicles was related to plasma-membrane adhesion rather than to internalized structures, as indicated by the bright signals present on plasma membrane near the edge of the cells (Figure 3b, left column). Thus, almost no internalization occurs in the absence of a targeting unit. Conversely, V40-DW4 shows a punctate pattern typical of receptor-mediated endocytosis, as expected on the basis of the known internalization pathway of the targeting aptamer (Figure 3b, right column). Note that non-stealth liposomes Plain-Lipo were readily internalized in cells, most likely owing to aspecific interaction with proteins and lipids at plasma membrane level (data not shown). Overall, these results demonstrate that the present stealth architecture completely abolishes aspecific uptake, allowing more efficient and selective targeting by surface units. Cells remained viable after internalization of V40-DW4, thus indicating the scarce cytotoxicity of the structures.


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Figure 3. Flow cytometry analysis and confocal microscopy imaging of MiA PaCa cells treated with V40 and V40-DW4 vesicles. A: Flow cytometry analysis of MiA-PaCa2 cells treated with fluorescently labeled V40 and V40-DW4; B: confocal microscopy images of MiA-PaCa2 cells treated with fluorescently labeled V40 (left column) and V40-DW4 (right column) Finally, we evaluated the effectiveness of V40-DW4 as delivery platform. To this end, we selected doxorubicin (Dox) as therapeutic payload. Dox has been extensively used in cancer



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therapy for decades and represents a well-assessed benchmark to compare and validate new therapeutic platforms.3 It is known that Dox encapsulation efficiency in lipid based vesicles is generally unsatisfactory if performed using spontaneous internalization during vesicle hydration. Thus, we decided to adopt an “active loading” strategy based on ammonium sulphate gradients.35 Briefly, we hydrated V40-Mal and V40 (untargeted control) in the presence of a 300 mM solution of ammonium sulphate; next, the external buffer was exchanged with PBS and doxorubicin was added to the solution. When this protocol is applied to standard liposomes, Dox loading is performed for prolonged periods at low temperatures (4-8 oC). In our case, however, internalization required considerably higher (60 o

C) temperature and shorter times leading to Dox loaded vesicles Dox-V40-Mal and Dox-

V40 in less than one hour. Loading efficiency, measured by spectrophotometric evaluation of encapsulated Dox according to established protocols36 was in both cases around 30%. It is tempting to assume that sensibly higher loading efficiencies could be easily achieved by extending incubation time; however, we decided to maintain such short incubation times to avoid thermal degradation or hydrolysis of the sensitive Dox payload and of maleimide groups. Interestingly, the loading temperature is close to the degradation temperature of V40 (Tab. 1), thus suggesting a critical role of lipid mobility and phase in the permeability of the vesicle to Dox. Notably, size and polydispersity index of Dox-V40-Mal and Dox-V40 did not change significantly during the process (Table S1), indicating that, even though membranes are permeable to Dox in these conditions, the overall mechanical stability of the vesicles is unaltered upon heating near their degradation point. As already mentioned, V40-DW4 are internalized by a receptor-mediated endocytic pathway. During this process, the nanoparticle experiences drastic changes in its surrounding environment, such as a significant lowering of pH that can attain values of 4.5 in lysosomes, and an increasingly reducing environment. The structure of L1 contains several glutamate


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residues, which are partially protonated in mildly acidic physiological conditions. Thus, we envisaged that pH could play a critical role in modulating the stability of V40, and this in turn could influence Dox release rate. The possibility to accelerate release Dox in environments with acidic pH could positively affect therapeutic efficiency of Dox-V40, allowing pHtriggered release at endolysosomal level. Thus, we evaluated the extent of Dox release in solution at pH varying from 7.4 (physiological conditions) to 4.5 (typical lysosome pH value). We observed that Dox release increases significantly at acidic pH, compared to what observed in neutral conditions (Figure 4a). Interestingly, Dox release at pH 4.5 is more than 60% greater compared with what observed in the control at pH 7.4. The observed leakage can be related to the destabilization effect conferred by glutamate protonation –and hence increase in vesicle net charge- at acidic pH and is in agreement with the reported pKa of glutamate side chain of 4.5.37 Overall, these results suggest that extensive Dox release should occur along the endocytic pathway upon internalization. The possibility to perform pHtriggered release confers a second control level triggered after selective aptamer-mediated internalization, and further supports the use of Dox-V40-DW4 as a delivery agent. To test this hypothesis, we finally sought to evaluate the cytotoxicity of Dox-V40 and Dox-V40DW4 towards MiA PaCa cells.



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Figure 4. a) Fluorescence increase of Doxorubicin released from vesicles upon incubation at different pH (pH 7.4=1); b) cell viability (72h) upon 30 minutes incubation of MiA PaCa cells with Dox-V40 and Dox-V40-DW4 We monitored cell viability after short (30 minutes) incubation with Dox-V40 and Dox-V40DW4. Longer incubation times were not necessary, since adhesion and internalization of DW4 usually occurs within 30 minutes.31 The extracellular medium was then changed to avoid any aspecific contribution due to Dox leakage from the vesicles. In keeping with the observed selective internalization shown by empty vectors (Figure 3), we observed that DoxV40-DW4 induces dose-dependent cell mortality at 48h (data not shown) and 72h upon administration (Figure 4b). Conversely, untargeted Dox-V40 was not harmful to the cells, except for a small cytotoxic effect at high concentration. Likely, this could be due to a minor contribution of aspecific internalization, such as membrane recycling after loose binding of Dox-V40 to the membrane.


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Conclusions In conclusion, we presented a new, nanostructured vesicular architecture, composed of both lipids and lipopeptides, which presents very interesting properties in terms of stability, ease of preparation, and functionalization flexibility. We demonstrated that optimal stability is obtained in a relatively narrow range of lipopeptide/lipid ratio and that at this composition virtually no serum protein adsorption is observed even after prolonged incubation in pure serum. Notably, different peptide motifs can be placed on the surface of the vesicle by simple co-formulation of different species, further widening the application range of this platform. Furthermore, the simple assembly process allows insertion of different homing sequences thus allowing preparation of multifunctional vesicles. These nanostructures can perform efficient targeted delivery of therapeutic agents without affecting their intrinsic cytotoxic effects, but allow targeted delivery of the therapeutic payload. Most importantly, the proposed nanostructures outperform benchmark stealth PEGylated liposomes in terms of serum adsorption and thermal stability. This feature shows exciting opportunities towards the realization of a new generation of stealth vesicles that are free from PEG or other unnatural polymers and suitable for repeated administration in living organisms. Further studies are in progress to evaluate stability and biodistribution in vivo, as well as possibility to deliver more complex molecular payloads in specific subcellular compartments.

Experimental Section Materials All chemicals were purchased from Sigma Aldrich unless otherwise specified, and were used as received. Dynamic Light Scattering (DLS) measurements



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DLS measurements were performed at 25°C in a 50-µL quartz cuvette on a Zetasizer nano ZS DLS (Malvern Instrument) following the manufacturer’s instructions. Water solutions of vesicles were analyzed with a single scattering angle of 90°. Each value reported is the average of five consecutive measurements. Peptide synthesis and purification All peptides were prepared by solid-phase synthesis using Fmoc chemistry on an automatic Liberty Blue Peptide Synthesizer with an integrated microwave system (CEM, North Carolina, USA). Standard conditions suggested by the producer were used thorough the synthesis. Attachment of palmitoyl chains was performed as follows: a di-Fmoc lysine was used in the last coupling step to introduce a branching point. Next, two deprotection steps were performed, followed by two coupling steps using palmitic acid. Other conditions specific for the two peptides- are detailed below. In both cases, the deprotected peptide was precipitated with cold diethyl ether after removing in vacuo most of the cleavage cocktail. HPLC analyses and purifications were performed on a Dionex Ultimate 3000 HPLC system or on a Shimadzu Nexera HPLC system. Crude peptides were purified by RP-HPLC on a Jupiter Proteo 90 Å column (4µm, 250×10 mm; Phenomenex) using water:TFA 100:0.01 v/v (eluent A) and acetonitrile:water:TFA 95:5:0.01 v/v (eluent B) as mobile phase. The identity of purified product was confirmed by electrospray mass spectroscopy, using an API3200QTRAP Hybrid Triple Quadrupole/Linear Ion Trap (ABSciex, Foster City, California, USA). Synthesis of L1 Peptide L1 was synthesized with the following experimental conditions: Resin: Rink-amide, loading 0.46 mmol/g (Bachem)


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Cleavage conditions: cleavage cocktail was composed by TFA/ Tri(isopropylsilane)/ H2O/ Ethanedithiol/ thioanisole 93/2/2/2/1; cleavage was performed according to manufacturer conditions (30 minutes at 30oC under microwave irradiation). Synthesis of L1-Mal Peptide L1-Mal was synthesized in two steps: synthesis of the fully protected peptide and conjugation with aminoethyl maleimide. Synthesis of the protected peptide was performed using the following experimental conditions: Resin: Preloaded (Glu) SASRIN resin, loading 0.46 mmol/g (Bachem) Cleavage of the protected peptide: SASRIN resin containing the protected peptide was thoroughly washed with DCM, then treated with 2 mL of 1% TFA in DCM for 5 minutes. The surnatant was collected by suction in a vessel containing pyridine in methanol (1/10 v/v, 2mL total volume) and the process was repeated six times, collecting each solution separately. Next, the fractions were analyses by LC-MS, and those containing the peptide were joined. The solvent was removed in vacuum, and the product purified by semipreparative HPLC. Conjugation

of

aminoethylmaleimide

to

protected

L1:

protected

peptide,

2-

aminoethylmaleimide hydrochloride (2 equivalents), triethylamine (2 equivalents), HOBt hydrate (1.1equivalent), and DIC (1.5 equivalents) were dissolved in anhydrous DMF and stirred during 5 hours. Reaction progress was monitored by HPLC. Then, the solvent was removed in vacuum, and 1 mL of TFA was added. The mixture was stirred for 4 hours, and the crude product was purified by semipreparative HPLC. Preparation of synthetic vesicles



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The appropriate lipid/lipopeptide mixture was inserted in a glass vial, and diluted with dimethyl sulfoxide (1mL/100ug of total lipid/lipopeptide). The solution was frozen in liquid nitrogen and freeze –dried. The resulting solid could be stored at -20 oC for several months without significant alterations. The solid was diluted with a PBS or ammonium sulfate (150mM) solution (2mL/mg of organic material), and mixed at 60oC and 500 rpm for 30 minutes. Next, the solution was extruded 21 times through a polycarbonate membrane (pore size: 200nm), keeping the solution at 60 oC with a heated liposomal extruder. The extruded vesicles were dialyzed once against PBS to remove non-encapsulated L1, and stored at 4 oC until use. Although the vesicles were usually stable for several weeks, they were used within a few days in all experiments. Quantification of L1 and DSPC in the vesicle A solution of V40 was diluted (1:10) in a 90/10 isopropanol/water mixture containing 0.1% formic acid, and mixed at room temperature for 15 minutes. Then, a known amount of solution (5µL) was injected in a Phenomenex Kinetex C8 column (3x150mm), and a gradient was performed with the following conditions: Phase A: H2O/MeOH/iPrOH/formic acid 55/40/5/0.1%, Phase B: MeOH/iPrOH/formic acid 95/5/0.1%, Linear gradient from 100%A to 100%B in 30 minutes, flow 1mL/min. Two MRM traces were recorded for each analyte (1013.4/322 and 1013/605 for L1, 790.7/184 and 790.7/524.3 for DSPC), with the former used for quantitation and the latter for confirmation purposes in each case. Concentration of both analytes was calculated upon comparison with a standard calibration curve. Evaluation of serum protein adsorption of vesicles


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The appropriate vesicle solution was diluted (1:50) in pre-heated, pure FBS. The solution was immediately placed at 37oC and size measurements were performed every 10 minutes during an 8 hour period. A sample of pure FBS from the same lot was analyzed to identify peaks present in the DLS measurement arising from serum. Each measurement was repeated in triplicate. Evaluation of vesicle thermal stability The appropriate vesicle solution was diluted (1:50) in PBS at 25oC. The solution was placed in the DLS thermostatized at the same temperature, and measurements were performed with increments of 5oC up to a maximum temperature of 65oC. In each step, the heating phase was followed by a 15 minutes equilibration. Each measurement was repeated in triplicate. Coupling of thiol-derivatized aptamer with L1-Mal Thiol-derivatized aptamer DW4 (sequence: [ThiC6]GCG TGT GCA CAC GGT CAC TTA GTA TCG CGG CGT TCT TTG GTT CCG CCC GG), with thiol functional group protected as disulfide, was treated with 25 equivalents of TCEP in degassed PBS for 2h. Then, the solution was quickly dialyzed on membranes with molecular weight cutoff 3 kDa. The resulting solution was added to a PBS solution of vesicles keeping the molar ration between L1-Mal and DW4 1:10. The solution was stirred at room temperature for 3 hours, and extensively (3 times) dialyzed at 4oC on membranes with molecular weight cutoff 50kDa and stored at 4oC until the use. Quantification cell uptake of targeted and untargeted vesicles in MiA PaCa cells by flow cytometry Mia Paca-2 cells were seeded in a 12-well plate at a concentration of 1x105 cells/well to reach 80-90% of confluency after 24h. For the experiment cells were washed once with PBS



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solution and were maintained on starvation for 1h in DMEM without serum at 37°C. Different liposome preparations were used to treat cells at a concentration of 2µM for 45 minutes at 37°C. After that each well was washed twice with PBS solution, trypsinized and washed again. Samples were filtered and immediately analyzed with S3e Cell Sorter (BioRad). Final results were elaborated with FlowJo Software. Loading of vesicles with Doxorubicin The vesicles extruded in 300 mM ammonium phosphate (pH 7.2) were mixed with a 10 mg/mL solution of Doxorubicin in water, using a 3/1 Dox/vesicle w/w ratio. The solution was stirred at 60oC for 30 minutes, cooled and dialyzed at 4oC on a 50 kDa membrane. Concentration of Dox in the vesicles was evaluated spectrophotometrically by diluting a known amount of vesicle solution in a 90/10 isopropanol/water mixture containing 0.075M HCl, according to a reported protocol.36 Measurement of doxorubicin release in cuvette Dox-loaded nanoparticles were diluted in solutions (citrate/phosphate 20/60 mM) buffered at different pH from 7.4 to 4.64, keeping constant nanoparticle concentration in the final solution. The system was equilibrated at 25 oC and fluorescence emission was measured with

λexc =480nm and λem= 500-600nm immediately after addition and after 15 minutes. Fluorescence increase was normalized on the basis of what observed at pH 7.4. Cell culture Human pancreatic carcinoma cells (MIA PaCA-2) were purchased from the American Type Culture Collection (ATCC). This cell line was growth using a previously reported protocol.31


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Confocal imaging of cells Cells were imaged using a Leica TCS SP5 SMD inverted confocal microscope (Leica Microsystems AG) interfaced with Ar laser for excitation at 488nm. Glass bottom Petri dishes containing cells were mounted in a thermostatized chamber at 37°C (Leica Microsystems) and viewed with a 63x 1.2 NA water immersion objective or 40x 1.5 NA oil immersion objective (Leica Microsystems). The pinhole aperture was set to 1.0 Airy. All data collected were analyzed by ImageJ software version 1.44o.

Assessment of cellular uptake by confocal microscopy Internalization, cellular uptake, and release of fluorescent payload assays were performed as follows. MIA PaCa-2 cells were seeded 24 h before the experiment in WillCo dishes to reach 80-90% confluence. Standard conditions for incubation consisted in 30 min incubation at 37°C, 5% CO2 in DMEM. After incubation, cells were washed three times with PBS, fresh serum-containing medium was added and the sample was imaged by confocal microscopy.

WST-8 Cell viability assay Cytotoxicity of Dox-Apt-NPs complexes was evaluated by using a tetrazolium salt, 2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H

tetrazolium,

monosodium salt (WST-8) assay. MIA PaCA-2 cells (1x104 cells per well) were seeded in 96-well plates. After culture for 24 hours, the cells were incubated with a 2% serumcontaining medium in the presence of vesicles at Dox concentration ranging from 0 to 10µM in DMEM (100uL) for 30 minutes at 37 oC. After the incubation, the medium was removed and cells were washed twice with PBS and kept in fresh DMEM. For each experimental time



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point cells were incubated with WST-8 reagent (20 µL) and 2% serum-containing medium (80 µL) for 2 hours. Absorbance (450nm) was measured using a microplate reader (Glomax Discovery, Promega). The percentage of cell viability was determined by comparing drugtreated cells with the untreated cells (100% viability). Data represent the average of three independent experiments. Error bars represent the SD from three independent experiments. AUTHOR INFORMATION Corresponding Author *Giovanni Signore, e-mail: [email protected], Laboratorio NEST, piazza san Silvestro, 12, 56127 Pisa Italy Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Supporting Information Available Complete characterization of Dox loaded vesicles (Dox-V40-Mal and Dox-V40). Acknowledgments The authors thank Dr. Valentina Cappello for her precious help in the characterization of nanoparticles.

REFERENCES (1) Danhier, F., Feron, O., and Préat, V. (2010) To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Control. Release 148, 135–46.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(2) Vittorio, O., Voliani, V., Faraci, P., Karmakar, B., Iemma, F., Hampel, S., Kavallaris, M., and Cirillo, G. (2014) Magnetic catechin–dextran conjugate as targeted therapeutic for pancreatic tumour cells. J. Drug Target. 5, 408-15 (3) Voliani, V., Signore, G., Vittorio, O., Faraci, P., Luin, S., Peréz-Prieto, J., and Beltram, F. (2013) Cancer phototherapy in living cells by multiphoton release of doxorubicin from gold nanospheres. J. Mater. Chem. B 1, 4225. (4) Cheng, J., Teply, B. A., Sherifi, I., Sung, J., Luther, G., Gu, F. X., Levy-Nissenbaum, E., Radovic-Moreno, A. F., Langer, R., and Farokhzad, O. C. (2007) Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery. Biomaterials 28, 869–76. (5) Costantino, L., Gandolfi, F., Tosi, G., Rivasi, F., Vandelli, M. A., and Forni, F. (2005) Peptide-derivatized biodegradable nanoparticles able to cross the blood-brain barrier. J. Control. Release 108, 84–96. (6) Barenholz, Y. (2012) Doxil- The first FDA-approved nano-drug: Lessons learned. J. Control. Release 160, 117–134. (7) Yabbarov, N. G., Posypanova, G. a, Vorontsov, E. a, Obydenny, S. I., and Severin, E. S. (2013) A new system for targeted delivery of doxorubicin into tumor cells. J. Control. Release 168, 135–41. (8) Chang, Y., Meng, X., Zhao, Y., Li, K., Zhao, B., Zhu, M., Li, Y., Chen, X., and Wang, J. (2011) Novel water-soluble and pH-responsive anticancer drug nanocarriers: DoxorubicinPAMAM dendrimer conjugates attached to superparamagnetic iron oxide nanoparticles (IONPs). J. Colloid Interface Sci. 363, 403–409. (9) Porciani, D., Tedeschi, L., Marchetti, L., Citti, L., Piazza, V., Beltram, F., and Signore, G. (2015) Aptamer-Mediated Codelivery of Doxorubicin and NF-κB Decoy Enhances Chemosensitivity of Pancreatic Tumor Cells. Mol. Ther. Acids 4, e235. (10) Li, Shyh-Dar and Huang, L. (2010) Stealth Nanoparticles: High Density but Sheddable PEG is a Key for Tumor Targeting. J. Control. Release 145, 178–181. (11) Salvati, A., Pitek, A. S., Monopoli, M. P., Prapainop, K., Bombelli, F. B., Hristov, D. R.,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Kelly, P. M., Åberg, C., Mahon, E., and Dawson, K. a. (2013) Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nat. Nanotechnol. 8, 137–43. (12) Monopoli, M. P., Walczyk, D., Campbell, A., Elia, G., Lynch, I., Bombelli, F. B., Dawson, K. a., Baldelli Bombelli, F., and Dawson, K. a. (2011) Physical-chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles. J. Am. Chem. Soc. 133, 2525–2534. (13) Santi, M., Maccari, G., Mereghetti, P., Voliani, V., Rocchiccioli, S., Ucciferri, N., Luin, S., and Signore, G. (2016) Rational Design of a Transferrin-Binding Peptide Sequence Tailored

to

Targeted

Nanoparticle

Internalization.

Bioconjug.

Chem.

acs.bioconjchem.6b00611. (14) Papahadjopoulos, D., Allen, T. M., Gabizon, a, Mayhew, E., Matthay, K., Huang, S. K., Lee, K. D., Woodle, M. C., Lasic, D. D., and Redemann, C. (1991) Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. U. S. A. 88, 11460–11464. (15) Immordino, M. L., Dosio, F., and Cattel, L. (2006) Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. Int. J. Nanomedicine 1, 297–315. (16) Stefanick, J. F., Ashley, J. D., Kiziltepe, T., and Bilgicer, B. (2013) A systematic analysis of peptide linker length and liposomal polyethylene glycol coating on cellular uptake of peptide-targeted liposomes. ACS Nano 7, 2935–2947. (17) Wang, X., Ishida, T., and Kiwada, H. (2007) Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J. Control. Release 119, 236–244. (18) Ishida, T., and Kiwada, H. (2008) Accelerated blood clearance (ABC) phenomenon upon repeated injection of PEGylated liposomes. Int. J. Pharm. 354, 56–62. (19) Xu, H., Wang, K. Q., Deng, Y. H., and Chen, D. W. (2010) Effects of cleavable PEGcholesterol derivatives on the accelerated blood clearance of PEGylated liposomes. Biomaterials 31, 4757–4763.


Page 27 of 35

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(20) Kowalczyk, B., Lagzi, I., and Grzybowski, B. a. (2011) Nanoseparations: Strategies for size and/or shape-selective purification of nanoparticles. Curr. Opin. Colloid Interface Sci. 16, 135–148. (21) Li, Y., Liu, R., Yang, J., Shi, Y., Ma, G., Zhang, Z., and Zhang, X. (2015) Enhanced retention and anti-tumor efficacy of liposomes by changing their cellular uptake and pharmacokinetics behavior. Biomaterials 41, 1–14. (22) Parodi, A., Quattrocchi, N., van de Ven, A. L., Chiappini, C., Evangelopoulos, M., Martinez, J. O., Brown, B. S., Khaled, S. Z., Yazdi, I. K., Enzo, M. V., et al. (2013) Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions. Nat. Nanotechnol. 8, 61–8. (23) Jang, S. C., Kim, O. Y., Yoon, C. M., Choi, D.-S., Roh, T.-Y., Park, J., Nilsson, J., Lötvall, J., Kim, Y.-K., and Gho, Y. S. (2014) Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors. ACS Nano 8, 1073. (24) El-Andaloussi, S., Lee, Y., Lakhal-Littleton, S., Li, J., Seow, Y., Gardiner, C., AlvarezErviti, L., Sargent, I. L., and Wood, M. J. a. (2012) Exosome-mediated delivery of siRNA in vitro and in vivo. Nat. Protoc. 7, 2112–26. (25) Nowinski, A. K., White, A. D., Keefe, A. J., and Jiang, S. (2014) Biologically inspired stealth peptide-capped gold nanoparticles. Langmuir 30, 1864–70. (26) Nowinski, a K., Sun, F., White, a D., Keefe, a J., and Jiang, S. (2012) Sequence, structure, and function of peptide self-assembled monolayers. J Am Chem Soc 134, 6000– 6005. (27) Lu, T., Wang, Z., Ma, Y., Zhang, Y., and Chen, T. (2012) Influence of polymer size, liposomal composition, surface charge, and temperature on the permeability of pH-sensitive liposomes containing lipid-anchored poly(2-ethylacrylic acid). Int. J. Nanomedicine 7, 4917– 4926. (28) Shroff, K., Pearce, T. R., and Kokkoli, E. (2012) Peptide targeted lipid nanoparticles for anticancer drug delivery. Adv. Mater. 24, 3803–3822. (29) Liang, M. T., Davies, N. M., and Toth, I. (2005) Encapsulation of lipopeptides within



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

liposomes: effect of number of lipid chains, chain length and method of liposome preparation. Int. J. Pharm. 301, 247–54. (30) Sokolova, V., Ludwig, A.-K., Hornung, S., Rotan, O., Horn, P. a, Epple, M., and Giebel, B. (2011) Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf. B. Biointerfaces 87, 146–50. (31) Porciani, D., Signore, G., Marchetti, L., Mereghetti, P., Nifosì, R., and Beltram, F. (2014) Two Interconvertible Folds Modulate the Activity of a DNA Aptamer Against Transferrin Receptor. Mol. Ther. Acids 3, e144. (32) Chen, C. B., Dellamaggiore, K. R., Ouellette, C. P., Sedano, C. D., Lizadjohry, M., Chernis, G. a, Gonzales, M., Baltasar, F. E., Fan, A. L., Myerowitz, R., et al. (2008) Aptamer-based endocytosis of a lysosomal enzyme. Proc. Natl. Acad. Sci. U. S. A. 105, 15908–13. (33) Deng, K., Hou, Z., Li, X., Li, C., Zhang, Y., Deng, X., Cheng, Z., and Lin, J. (2015) Aptamer-mediated up-conversion core/MOF shell nanocomposites for targeted drug delivery and cell imaging. Sci. Rep. 5, 7851. (34) Tassa, C., Duffner, J. L., Lewis, T. A., Weissleder, R., Schreiber, S. L., Koehler, A. N., and Shaw, S. Y. (2010) Binding affinity and kinetic analysis of targeted small moleculemodified nanoparticles. Bioconjug. Chem. 21, 14–9. (35) Fritze, A., Hens, F., Kimpfler, A., Schubert, R., and Peschka-Süss, R. (2006) Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient. Biochim. Biophys. Acta - Biomembr. 1758, 1633–1640. (36) Eliaz, R. E., and Szoka, J. (2001) Liposome-encapsulated doxorubicin targeted to CD44: A strategy to kill CD44-overexpressing tumor cells. Cancer Res. 61, 2592–2601. (37) Nozaki, Y., and Tanford, C. (1967) Intrinsic dissociation constants of aspartyl and glutamyl carboxyl groups. J. Biol. Chem. 242, 4731–4735.


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Table of Contents Graphic and Synopsis



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Scheme 1. Structure of stealth lipopeptide L1 56x19mm (300 x 300 DPI)


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Figure 1. diameter increase shown by different vesicles after incubation for 500 minutes in pure serum at 37oC, with respect to starting diameter. Plain-Lipo are completely degraded within 60 minutes, thus the reported measurement is performed after 50 minutes. 79x56mm (150 x 150 DPI)



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Scheme 2. synthesis of functionalizable lipopeptide L1-Mal. Asterisks indicate the presence of Boc or t-Bu protecting groups. 144x151mm (300 x 300 DPI)


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Figure 2. Physico-chemical properties of functionalizable, functionalized, and benchmark vesicles. 82x120mm (96 x 96 DPI)



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Figure 3. Flow cytometry analysis and confocal microscopy imaging of MiA PaCa cells treated with V40 and V40-DW4 vesicles. A: Flow cytometry analysis of MiA-PaCa2 cells treated with fluorescently labeled V40 and V40-DW4; B: confocal microscopy images of MiA-PaCa2 cells treated with fluorescently labeled V40 (left column) and V40-DW4 (right column) 102x177mm (96 x 96 DPI)


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Figure 4. a) Fluorescence increase of Doxorubicin released from vesicles upon incubation at different pH (pH 7.4=1); b) cell viability (72h) upon 30 minutes incubation of MiA PaCa cells with Dox-V40 and Dox-V40DW4 84x124mm (96 x 96 DPI)


Peptide-Based Stealth Nanoparticles for Targeted ... - ACS Publications

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