Efficient Decoration of Nanoparticles Intended for Intracellular Drug

Jul 20, 2014 - We revealed that copper, the catalyst of the Click reactions, formed complexes with unreacted targeting residues and interfered with th...
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Efficient Decoration of Nanoparticles Intended for Intracellular Drug Targeting with Targeting Residues, As Revealed by a New Indirect Analytical Approach Veronika Kaplun and David Stepensky* Department of Clinical Biochemistry and Pharmacology, The Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

ABSTRACT: In our previous studies, we developed a nanodrug delivery system (nano-DDS) based on poly(lactic-co-glycolic acid) PLGA nanoparticles encapsulating antigenic peptide and fluorescent marker and 3-stage approach for its decoration with peptide targeting residues. The objectives of this study were (a) to develop methods for quantitative analysis of efficiency of individual conjugation steps and (b) to determine, based on these methods, the efficiency of our 3-stage approach of nano-DDS decoration. We prepared antigenic peptide-loaded PLGA-based nano-DDSs and sequentially decorated them with specific residues using carbodiimide and Click (azide−alkyne Huisgen cycloaddition using copper(I) catalysis) reactions. The extent of cargo encapsulation and release kinetics were analyzed using HPLC-based and colorimetric analytical methods. The efficiency of residue conjugation to the nano-DDSs was analyzed using FTIR spectroscopy and by quantifying the unreacted residues in the reaction mixture (i.e., by indirect analysis of reaction efficiencies). We revealed that copper, the catalyst of the Click reactions, formed complexes with unreacted targeting residues and interfered with the analysis of their conjugation efficiency. We used penicillamine (a chelator) to disrupt these complexes, and to recover the unreacted residues. Quantitative analysis revealed that 28,800−34,000 targeting residues (corresponding to 11−13 nm2 surface area per residue) had been conjugated to a single nanoDDS using our 3-stage decoration approach, which is much higher than previously reported conjugation efficiencies. We conclude that the applied analytical tools allow quantitative analysis of nano-DDSs and the efficiency of their conjugation with targeting residues. The 3-stage decoration approach resulted in dense conjugation of nano-DDSs with targeting residues. The present decoration and analytical approaches can be effectively applied to other types of delivery systems and other targeting residues. KEYWORDS: targeted drug delivery, intracellular targeting, nanoparticle formulations, conjugation of targeting residues, Click chemistry

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targets in specific organelles.1,2 Several types of nano-DDSs intended for intracellular drug targeting have been developed,1,2 and preferential delivery of the drug to the target organelles has been claimed in several studies (reviewed by Stepensky3). Targeting moieties that were used for this purpose included (1) peptide sequences that are recognized by the cytosolic transport systems of the host cell, such as endoplasmic

INTRODUCTION Targeted delivery of drugs to their site of action in the body is a topic of extensive research in recent years. Efficient targeting is expected to enhance the desired pharmacological effects of a drug and minimize its adverse effects. Therefore, it will result in safer use of pharmacological agents and will improve the efficiency of treatment of many diseases. Targeting of the drugs to their site of action can be performed on several levels: (1) to the specific organ or tissue, (2) to the specific cells, and (3) to the specific intracellular organelle(s). Intracellularly targeted drug delivery is a newer level of targeting that can be used for drugs that act on intracellular © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2906

April 6, 2014 May 27, 2014 July 20, 2014 July 20, 2014 dx.doi.org/10.1021/mp500253r | Mol. Pharmaceutics 2014, 11, 2906−2914

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Figure 1. Developed formulation for intracellularly targeted drug delivery. (A) The formulation was based on PLGA nanoparticles loaded with antigenic peptide and fluorescent marker. (B) The decoration of the nano-DDS surface was performed by stepwise conjugation of the branching peptide, linker, and targeting peptide.

(via covalent binding, or due to noncovalent adsorption; see above). The number of the targeting residues on the surface of the single nano-DDS and their spacing and conformation are very important parameters of the developed formulation that are expected to affect its interaction with the target cells.3 Understanding of the mechanisms and the rate-limiting steps of the nano-DDS endocytosis, intracellular trafficking, and targeting cannot be complete without quantitative analysis of the decoration efficiency of nano-DDS with targeting residues. Presently we sought to quantify in a detailed fashion the efficiency of nano-DDS decoration with targeting residues. Therefore, the specific objectives of this study were (a) to develop methods for quantitative analysis of efficiency of the individual decoration steps and (b) to apply these methods to determine the efficiency of decoration of nano-DDSs using the 3-stage approach that we have developed in our previous studies.

reticulum (ER) signal peptide or ER-retrieval sequence, nuclear localization signal (NLS), mitochondrial localization signal, etc.; and (2) peptide or nonpeptide molecules that preferentially interact with the membrane of the target organelle, e.g., mitochondriotropic arginine-rich peptides or positively charged compounds. Several approaches for decoration of nano-DDSs with specific targeting residues have been applied by different research groups.4 This decoration can be noncovalent due to electrostatic or hydrophobic interaction between the surface of the nano-DDSs and the targeting residues, or via avidin−biotin complex formation. Covalent binding of the targeting residues to the surface of nano-DDSs can be attained via carbodiimide or sulfhydryl reactions, Click reaction, and other conjugation approaches. Only a small number of publications analyzed and reported the efficiency of decoration of nano-DDSs with targeting residues or contained quantitative data that can be used to calculate it (see Discussion). Moreover, based on the data reported in these studies, it is not clear whether the residues were indeed covalently conjugated to the nano-DDSs or became noncovalently adsorbed to them. In our previous studies, we developed a nano-DDS based on PLGA nanoparticles encapsulating antigenic peptide and fluorescent marker and decorated with peptidic targeting residues (see Figure 1A).5 These targeting residues were attached to the nano-DDS using a 3-stage decoration approach based on carbodiimide and Click (azide−alkyne Huisgen cycloaddition using copper(I) catalysis) reactions (see Figure 1B). We characterized the size and morphology of the developed DDS, and we analyzed their cellular uptake by dendritic cells in vitro, intracellular trafficking, and crosspresentation efficiency of the encapsulated antigenic peptide.5 However, in our previous studies (like in the majority of other studies in the nano-DDS research field, see above) we did not analyze the efficiency of nano-DDS decoration with the specific residues at the individual decoration steps, and it was not clear how many targeting residues actually became attached to the single nano-DDS at the end of the decoration procedure

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EXPERIMENTAL SECTION Preparation and characterization of the nano-DDS was performed using the same methods that we applied in our previous studies,5 with minor modifications. On the other hand, new experimental approaches were now devised to determine the efficiency of conjugation of the specific residues to the nano-DDS following the individual decoration steps (see below). Materials. Poly(DL-lactide-co-glycolide) polymer (PLGA, 50:50 monomer ratio, with free carboxylic end groups, MW 31−58 kDa) was from LACTEL (DURECT Corp., USA). Antigenic peptide (SIINFEKL), branching peptide (ADGADGADG), and propiolic acid conjugated ER-targeting (AAKDEL, AAKKYL) and nucleus-targeting (PKKKRKVKAA) peptides were synthesized by GL Biochem, China. Bovine serum albumin labeled with fluorescein isothiocyanate (BSAFITC), poly(ethylene-alt-maleic anhydride) (PEMA), N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid (MES), and penicillamine were from 2907

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1000 nm at the National Institute of Biotechnology (BenGurion University of the Negev, Beer-Sheva, Israel). Success of the individual decoration steps was qualitatively assessed by Fourier transform infrared analysis (FTIR). Samples of the nano-DDSs were placed on ZnSe crystals and analyzed using an IR Scope II microscope equipped with EQUINOX 55/S FTIR spectrometer (Bruker, Billerica, MA, USA). Three spectra (wavelength range of 600−4000 cm−1) were collected from the nano-DDSs (before and after the individual decoration stages) and averaged. Analysis of the Efficiency of the Individual Decoration Steps. Several processes can interfere with the conjugation of the residues to the nano-DDS at each decoration step. To determine the efficiency of these processes, we prepared control samples (see Table 1) and quantified the content of the

Sigma-Aldrich (Rehovot, Israel). Isopropanol, dichloromethane, and other analytical grade solvents were from BioLab, Israel. All other reagents were of analytical grade. Nanodrug Delivery System Preparation. The nanoDDS was prepared by a double emulsion technique (w/o/w emulsion) according to commonly applied protocols and experimental settings.6,7 A solution of BSA-FITC and SIINFEKL in PBS (2 mg/mL of each component, 200 μL) was added to a solution of PLGA in dichloromethane (100 mg/mL, 2 mL), and sonication was performed using a Vibracell CVX 130 probe sonicator (Sonics, Newtown, CT, USA) set at 50 W energy output with probe No. 3 for 2 min on ice. To the resulting o/w emulsion was added an aqueous solution of PEMA (20 mg/mL, saturated with DCM, 5 mL), and a w/o/w emulsion was generated by sonication for 5 min under the same conditions. The generated emulsion was transferred to an aqueous solution of PEMA (3 mg/mL, 50 mL) and was vigorously stirred for 5 min. After that, an aqueous solution of PEMA with isopropanol (9:1, v:v, 50 mL) was added and the formulation was stirred for 3 h in a chemical safety cabinet for complete evaporation of the organic solvents. The nano-DDSs were sedimented by centrifugation (at 3200g), washed, resuspended in doubly deionized water, and lyophilized using FreeZone 2.5 Plus Lyophilizer (Labconco, Kansas City, MO, USA). Decoration of the Nano-DDS with Branching, Linker, and Targeting Residues. In the first stage, the branching peptide was conjugated to the nano-DDS’s surface using carbodiimide reaction. The nano-DDS (50 mg) was resuspended in MES buffer (100 mM, pH 5.8) and underwent reaction with EDC and NHS (10 and 5 M, respectively) for 30 min at room temperature. The nano-DDS with activated carboxylic groups was washed, was resuspended in borate buffer (200 mM, pH 8.5), underwent reaction with the branching peptide (10 mM) for 2 h at room temperature, and was washed with PBS (100 mM, pH 7.4) afterward. In the next stage, the linker (3-azidopropylamine, 10 mM) was conjugated to the nano-DDS decorated with branching peptide using the carbodiimide reaction and the same synthesis conditions. The linker was synthesized from sodium azide and 3-chloropropylamine according to the procedure described by Jiang et al.8 In the last stage, the targeting or control peptides were conjugated to the linker using the Click reaction. The nanoDDSs decorated with the branching peptide and linker (15 mg) were resuspended in an aqueous solution of copper sulfate and sodium ascorbate (8 mM and 50 mM, respectively), propiolic acid N-conjugated ER-targeting or control (scrambled) peptide (5 mM) was added, and the suspension was incubated for 3 h at room temperature with constant stirring. The nano-DDSs were sedimented by centrifugation, washed, resuspended in doubly deionized water, and lyophilized. Analysis of the Nano-DDSs. The nano-DDSs’ morphology was studied by scanning electron microscopy (SEM). Sample of the lyophilized formulations were placed on carbon adhesive tape, coated with gold, and imaged using a Quanta 200 scanning electron microscope (Hillsbro, OR, USA) at the Institute of Applied Research (Ben-Gurion University of the Negev, Beer-Sheva, Israel). The nano-DDSs’ size and ζpotential were measured (in 1 mM HEPES buffer, pH = 7.4) using a ZetaPlus instrument (Brookhaven Instruments Corporation Ltd., Holtsville, NY, USA) at the range of 10−

Table 1. Experimental Setup That Was Applied for Analysis of the Processes That Took Place during the Drug Delivery System Decoration Reactions sample

residue

nano-DDS

catalysts

incubation

initial degradation adsorption sedimentation conjugation

+ + + + +

− − + − +

− − − + +

− + + + +

residue in the supernatant of these samples at the end of incubation. Lower residue concentration in the “degradation” vs “initial” sample reflects residue degradation during the incubation. Lower residue concentration in the “sedimentation” vs “degradation” sample reflects residue sedimentation during the incubation or adsorption to the walls of the tube, etc. Complexation of the catalysts of the Click reaction (Cu) with peptide targeting residues interfered with the analysis of the nano-DDS decoration with these residues. We reversed this complexation using penicillamine, a chelator of copper ions. We determined the ability of the penicillamine to disrupt the copper−peptide interaction and to recover the free peptide for different targeting residues and at different concentrations of peniciallamine (0−400 mM; the concentrations of the peptides and of the catalysts were kept the same as described in the previous section). The optimal concentration of penicillamine was identified in these experiments, and a corresponding amount of penicillamine was added to the catalyst-containing samples (see Table 1) at the end of incubation to recover the free peptide prior to HPLC analysis of its content in the supernatants. HPLC Analysis of the Supernatants. Supernatants of the individual samples were analyzed using a high-performance liquid chromatography (HPLC) method utilizing a Waters liquid chromatography system (Waters Alliance 2695 system with 996 diode array detector). A Phenomenex Luna C18 column (5 μm, 150 × 4.6 mm) was used for the LC separation. The mobile phase consisted of 0.1% trifluoroacetic acid and acetonitrile with flow rate set to 1.0 mL/min. A gradient was applied by increasing the acetonitrile concentration from 3% at the first 5 min to 30% at 25 min, 100% at 27 min (for 2 min, to wash the column), and then back to 3% concentration, which was kept constant until the end of the run time at 32 min. The column was kept at 30 °C, the injection volume was 10 μL, and detection at 220 nm was applied. Concentrations of the individual peptides in the analyzed solutions were calculated 2908

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Figure 2. FTIR spectrum of the drug delivery system prior to and after the individual conjugation steps (decoration with the branching peptide, linker, and AAKDEL targeting peptide). Outlined are the wavelengths that correspond to the individual functional groups.

Following individual decoration steps, characteristic changes of the FTIR spectra of the nano-DDSs were observed (see Figure 2). For instance, following decoration of nano-DDS with the branching peptide and the linker (3-azidopropylamine), prominent peaks appeared in the spectra that correspond to the carbonyl and azide groups (∼1630 cm−1 and ∼2100 cm−1, respectively). Decoration of the linker-conjugated nano-DDS with the AAKDEL targeting peptide decreased the area of the azide peak and led to appearance of a peak that corresponds to the triazole ring (∼1550 cm−1), which is expected to be formed from the azide and alkyne groups during the Click reaction. Thus, the obtained FTIR spectra are consistent with successful decoration of the nano-DDSs with branching peptide, linker, and the targeting peptides. However, these data are qualitative and do not reveal whether the specific residues became covalently attached to the surface of nanoDDS (or adsorbed to it) and to what extent. Interaction of the Catalysts of the Click Reaction (Cu) with the Targeting Peptides. We devised an experimental approach to determine the efficiency of the conjugation of the residues to the nano-DDS at each decoration step, and whether the processes can interfere with this conjugation (see Table 1). For the carbodiimide reactions (see Figure 1B), this approach worked well without need for modifications. However, we observed that, in the Click reactions, catalysts tended to form complexes with the targeting peptides that remained in the supernatant and interfered with the analysis of nano-DDStargeting peptide conjugation efficiency. Therefore, we studied the interaction of the catalysts with the individual targeting peptides, in the absence of nano-DDSs. Addition of catalysts to the solution of targeting peptide led to disappearance of the free peptide and formation of complexes that were eluted close to the void volume during the HPLC analysis (see representative chromatograms in Figure 3A). Addition of increasing concentrations of the penicillamine chelator disrupted these complexes and released the free peptide. The extent of complexation with catalysts depended on the amino acid sequence of the individual peptide (see Figure 3B), and the studied peptides had propensity to

from the obtained peak areas based on appropriate calibration curves. Calculation of the Efficiency of the Individual Conjugation Reactions. Based on the average diameter of the nano-DDSs (350 nm, which corresponds to a volume of 0.022 μm3, assuming a spherical shape of a nano-DDS and volume = (4/3)πr3) and density of PLGA (1.22 g/cm3), we estimated that the weight of a single nano-DDS was 27 × 10−15 g. Hence, we assumed that 1 mg of the prepared formulation contained approximately 1012 nano-DDSs (which is a conservative estimate of number of nano-DDSs per mg). Efficiency of the individual reactions was calculated based on this value, and on the amount of the residue that participated in the reaction (e.g., covalent conjugation of residues to the nanoDDSs was calculated from the difference between the “conjugation” vs “absorption” samples in the peptide content in the supernatant in the presence of penicillamine; see Table 1 and previous sections). The spacing of the conjugated targeting residues (surface area per residue) was calculated on the basis of the diameter and decoration efficiency of the DDSs, assuming that the DDSs had a spherical form (and the surface area = 4πr2). Statistical Analysis. The data are presented as means ± standard deviation (SD). Differences in the studied parameters between the experimental groups were analyzed using ANOVA with Tukey−Kramer post-test using InStat 3.0 software (GraphPad Software Inc.). p value less than 0.05 was termed significant.

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RESULTS Drug Delivery System Preparation and Characterization. We prepared spherical nano-DDS loaded with antigenic peptide (SIINFEKL) and fluorescent marker (BSAFITC). The properties of the formulation were very similar to those of the previously reported ones (Sneh-Edri et al.,5 data not shown). The average diameter of unconjugated nano-DDS was 350 nm, and the ζ-potential was −30 mV, which is consistent with the previously reported results. 2909

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Figure 3. Complex formation of the peptides with the catalysts of the Click reaction (Cu; in the absence of nano-DDS) and peptide release by penicillamine (the chelator). (A) Representative HPLC chromatograms for penicillamine-mediated recovery of one of the targeting peptides. (B) Dose−response of penicillamine-mediated recovery of the individual targeting peptides. Mean ± SD of 3 experiments. #: statistically different from both AAKDEL and PKKKRKVKAA peptides, p < 0.05. †: statistically different from PKKKRKVKAA peptide, p < 0.05.

form complexes with the catalysts in the following order: AAKKYL > PKKKRKVKAA > AAKDEL. Penicillamine concentrations of 100 mM or higher efficiently chelated the copper that was present in the Click reaction mixture and converted the majority of the peptides to their free form. Therefore, we chose to add penicillamine at a final concentration of 200 mM to the reaction mixture at the end of the decoration of the nano-DDS with targeting peptides (based on the Click reactions, see Figure 1B), to recover the free peptide from its complexes with catalysts and to allow quantitative HPLC-based analysis of reaction efficiency. Analysis of the Efficiency of the Individual Decoration Steps. We analyzed the efficiency of residue conjugation to the nano-DDS at each decoration step and to what extent the

processes can interfere with this conjugation (see Table 1). Representative chromatograms for one of the studied reactions are shown in Figure 4A. It can be see that, for this reaction, processes of peptide degradation and adsorption to the nanoDDS were minor. On the other hand, substantial amounts of the peptide in the reaction mixture underwent conjugation to the nano-DDS, or sedimented (became insoluble or adsorbed to the tube), or remained unreacted. Based on the obtained peak areas, the efficiency of decoration of the nano-DDS with the specific residues was determined for each decoration stage (absolute and relative efficiency of the individual processes, see Figure 4B and Table 2, respectively). Approximately 14,400 branching peptides, 30,600 linker molecules, and 28,800−34,000 targeting peptides 2910

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Figure 4. Analysis of the efficiency of the individual decoration steps of the drug delivery system. (A) Representative HPLC chromatograms for the analyzed samples of one of the targeting peptides. (B) Efficiencies of the processes that took place during the individual decoration steps (presented as number of residues per single nano-DDS that conjugated or adsorbed to its surface, sedimented in the reaction tube, degraded, or remained in the supernatant).

Table 2. Efficiencies of the Processes That Took Place during the Individual Decoration Steps (%, Average ± SD of 3 Experiments) residue

% unreacted

branching peptide linker

74.9 ± 1.7b 54.4 ± 6.0a

PKKKRKVKAA peptide AAKKYL peptide AAKDEL peptide

4.7 ± 4.2a,b,c 15.6 ± 3.0a,b,c 31.6 ± 9.1a,b

% degradation

% adsorption

Carbodiimide Reaction 1.8 ± 0.8 1.5 ± 1.2 Click Reaction 0 0 2.0 ± 4.0

0.7 ± 0.7b 9.0 ± 4.8a 0 0 2.3 ± 4.5

% sedimentation 0 0 56.7 ± 1.9a,b,c 52.9 ± 0.9a,b,c 41.0 ± 4.6a,b

% conjugation 22.8 ± 1.6b 35.1 ± 2.5a 38.1 ± 4.6a,c 31.5 ± 2.5a,c 23.5 ± 4.2b

a

Statistically different from branching peptide, p < 0.05. bStatistically different from linker, p < 0.05. cStatistically different from AAKDEL peptide, p < 0.05.

became conjugated to a single nano-DDS on the average (see Figure 4B), which indicates a high overall efficiency of the conjugation reactions. Approximately 23−35% of the residues were conjugated to the nano-DDS in the individual reactions under the applied

reaction conditions (see Table 2). For the carbodiimide reactions, a high fraction of the residues remained soluble in the reaction mixture. For the Click reactions, the soluble (unreacted) fraction was lower, and large amounts of the residues sedimented (became insoluble or adsorbed to the 2911

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Table 3. Efficiencies of Drug Delivery System Decoration with Peptidic Targeting Residues in Selected Recent Studies and in the Current Studya source

peptides per nanoDDS

surface area per peptide residue (nm2)d

carbodiimide followed by sulfo-SMCC amine-to-sulfhydryl cross-linking carbodiimide Click reaction

48 or 62b

46 or 236

2,500c 400b

90 135

carbodiimide

45,300c

4.3

PLGA-PEG NP-peptide (423 nm diameter) PLGA-PEG NP-peptide (210 nm diameter) PLGA NP-peptide (226 nm diameter)

carbodiimide maleimide − SH carbodiimide

3,550c 3,550c 443b

158 39 362

iron oxide NP-HER2/neu targeting affibodies (26 or 50 nm diameter) PLGA NP-peptide (350 nm diameter)

Click reaction

up to 72 or >100b as high as 29 or less than 79 28,800−34,000 11−13

formulation

Hoshino et al., 200431 quantum dot-peptide (30−60 nm diameter) Zhang et al., 200832 Lu et al., 200933 Chittasupho et al., 200923 Wang et al., 200934 Toti et al., 201035 Misra and Sahoo, 201036 Elias et al., 201330 Kaplun and Stepensky (this study)

PLGA-PEG NP-peptide (268 nm diameter) poly(TMCC-co-LA)-PEG NP-peptide (131 nm diameter) PLGA NP-peptide (248 nm diameter)

chemistry

3-stage reaction, 2 carbodiimide reactions followed by Click reaction

a

Decoration efficiency (i.e., amount of peptides that were attached to the surface of the nano-DDS) was either reported by the authors or calculated based on the data from the individual publications. bReported in the publication. cCalculated based on the publication data and the assumptions described in the Experimental Section. dCalculated based on the diameter and decoration efficiency of the DDSs, assuming that the DDSs had spherical form (and the surface area = 4πr2).

tube) during the course of the reaction. For both the carbodiimide and Click reactions, a small fraction of the residues underwent degradation or became adsorbed to the nano-DDS (except for the linker that had a tendency to adsorb/interact with the nano-DDS in absence of catalysts).

In this study, we revealed that copper forms complexes with peptides, and that the extent of this complexation depends on the structure of the peptide (see Figure 3B). We were able to disrupt these complexes and to release the unreacted peptide back into the reaction mixture using penicillamine chelating agent. Penicillamine is a clinically approved agent for the management of copper toxicity12,13 which binds copper and other heavy metals with high affinity. Under the conditions of our experiments, 100−200 mM penicillamine was sufficient to sequester the majority of copper in the experimental mixture (see Figure 3B). It is possible that the same effect can be also attained using EDTA, an additional chelator with high affinity to copper. In any case, sequestration of copper using a chelator allowed us to quantify the unreacted targeting residues in the reaction mix and to determine the efficiency of the covalent conjugation of these residues to the nano-DDSs in the individual decoration steps that were based on the Click reaction. It should be noted that, apart from interference with peptide conjugation to the nano-DDSs and its quantitative analysis, copper and especially copper-containing nanoparticles can be toxic to cells and biological systems.14,15 Therefore, copper content in nano-DDSs produced using Cu(I)-catalyzed Click reactions should be monitored and kept below a certain threshold. Alternatively, Cu-free Click chemistry can be used to decorate nano-DDSs with targeting residues16,17 in order to prevent copper toxicity and to eliminate the need for monitoring its content in the formulation. Decoration efficiency of nano-DDSs with targeting residues can be analyzed using numerous additional analytical approaches, including nuclear magnetic resonance spectroscopy (NMR), X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy (AES), combustion elemental analysis, and other techniques.18,19 These methods can provide important information regarding the surface composition of nano-DDS and the conformation of the surface residues (on the surface of nano-DDSs and/or in their deeper layers, depending on the specific analytical technique), but are less suited for quantitative analysis of the conjugated residues.18 Therefore, in our future studies, we plan to combine the

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DISCUSSION A New Approach for Decoration of Nano-DDSs and Analysis of Its Efficiency. In our previous studies, we devised a new approach for decoration of the nano-DDS with targeting residues.5 This approach is based on a 3-stage sequential decoration of the nano-DDSs with the branching peptide, linker, and the specific targeting residues. Overall, this approach, which is based on extensively characterized carbodiimide and Click reactions, is expected to be efficient, and indeed decoration of nano-DDSs using this approach affects their endocytosis and intracellular trafficking.5 In this manuscript we report new experimental approaches and findings that are based on the previous studies, are complementary to them, and allow more detailed characterization of the generated nano-DDSs. Specifically, we established an indirect approach (based on the analysis of the unreacted residues) to quantify the efficiency of the individual decoration steps. Setting up parallel reactions with selected components (see Table 1) allows quantitative analysis of the efficiencies of the individual processes that take place in the reaction mix (see Figure 4 and Table 2). During the course of our studies, we identified that peptidic targeting residues tend to form complexes with the catalysts of the Click reaction, and this complexation interferes with the analysis of the decoration efficiency. Click reaction is a widely used approach for decoration of nano-DDSs with targeting residues due to its high efficiency in aqueous and nonaqueous solvents.9,10 Cu(I) is a frequently used catalyst in this reaction (that originates from cupric sulfate and ascorbic acid, sources of monovalent copper during the course of the reaction). It has been reported previously that copper can form complexes with certain compounds and interferes with the quantification of the reaction products and their purity.11 2912

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for the extracellular events, endocytosis, and intracellular targeting of the nano-DDSs in vitro and in vivo are not clear. Overall, successful intracellular targeting in vivo is a very challenging task since it can be attained only after the drug has reached the target cells (e.g., for the nano-DDSs that we devisedtheir endocytosis of phagocytosis by the blood dendritic cells following intravenous dosing). Apparently, only a small fraction of a dose will be able to reach the intracellular target site following systemic nano-DDS administration, leading to limited targeting efficiencies (similar to low targeting efficiencies on the organ and cell levels of the currently available with DDS24−26). The effect of the surface density of the targeting residues on the nano-DDSs’ uptake by the cells in vitro has been studied by several research groups. In some studies, positive correlation has been found and nano-DDSs more densely decorated with the targeting residues were endocytosed more efficiently by the cells.27,28 On the other hand, in several other studies, intermediate surface density of the targeting residues provided statistically significant improvements in cell binding and endocytosis in comparison with higher and lower surface densities.29,30 This controversy apparently reflects the differences in the number of ligands that mediate the specific endocytosis process, their surface density and conformation that apparently affect their propensity to interact with receptors that mediate endocytosis, and differences in the size and surface charge of the formulations that were investigated in the individual studies. The quantitative relationship between the number of targeting residues and intracellular targeting efficiency is even more obscure.3 We plan to apply the analytical approach that we devised in this study (to quantify the efficiency of the nanoDDSs’ decoration with the targeting residues) to reveal this relationship and to determine the rate-limiting steps of the intracellular disposition of nano-DDSs. It should be noted that peptidic targeting residues on the surface of nano-DDSs are expected to undergo gradual degradation prior to and after their endocytosis. Therefore, it appears that the initial amount of the targeting residues on the surface of the nano-DDSs should be high in order to enhance the chances for efficient intracellular targeting following partial degradation of the residues during the course of the experiment. Additional analytical tools are needed to follow the time course of targeting residue degradation inside and outside the cells and to correct the experimental conclusions for the partial degradation of the targeting residues.

indirect analysis approach of nano-DDS decoration efficiency (that is reported in this study) with the above-mentioned analytical methods to reveal the effect on the surface density and conformation of the targeting residues on the nano-DDSs’ endocytosis and intracellular trafficking. Efficiency of the Conjugation Reactions. Using the indirect analytical approach that we devised (see above), we were able to determine the efficiency of the individual decoration steps (see Figure 4B and Table 2). It can be seen that the type of applied reaction (carbodiimide vs Click reaction) and the residue type (branching peptide, linker, or a specific targeting peptide) affected the efficiency of the individual decoration steps and of the individual processes that took place in the reaction mixture. The overall conjugation efficiency (i.e., covalent attachment of the residues to the nano-DDSs) was high with approximately 14,400 branching peptides, 30,600 linker molecules, and 28,800−34,000 targeting peptides that became conjugated to a single average nano-DDS (see Figure 4B). Thus, it appears that the relative ratio of the conjugated residues was 1:2:2 (for the branching peptides:linkers:targeting peptides, respectively), which is consistent with the conjugation strategy (see Figure 1B). However, the overall conjugation efficiency appears to be below the maximally possible values (that is expected to produce 1:3:3 ratios of the residues; see Figure 1B). Unfortunately, only a small number of publications analyze and report the efficiency of decoration of nano-DDSs with targeting residues or contain quantitative data that can be used to calculate it (see data from the selected publications and their analysis in Table 3). As can be seen, the estimated decoration efficiencies ranged from several dozens of residues to thousands or even dozens of thousands of residues per single nano-DDS. These values correspond to 4−356 nm2 nano-DDS surface area per individual targeting residue (see Table 3). However, it is not clear from the analyzed publications whether the residues were indeed conjugated to the nano-DDSs or became noncovalently adsorbed to them, and to what extent their conformation allows interaction with the target molecules. From the comparison of our results (Figure 4B) and the data presented in Table 3, we conclude that the decoration approach that was applied by us was efficient and that the generated nano-DDSs were densely conjugated with the targeting residues. Apparently, major factors that contributed to this high conjugation efficiency were (a) the high number of carboxyl groups on the surface of the nano-DDSs prior to their decorations (due to the use of PEMA surfactant20,21 instead of other stabilizing agents that do not contain carboxylic groups and mask the carboxylic groups that originate from the nanoDDS itself (e.g., in poly(vinyl alcohol), polysorbate-80, or poloxamer-188-stabilized PLGA-COOH nanoparticles22)), and (b) multiplication of the carboxylic groups on the surface of the nano-DDSs using the branching peptide with subsequent conjugation of the linker and targeting peptide (see Figure 1B). This conclusion appears to be supported by the fact that the only other densely decorated formulation (PLGA-COOH nanoparticles stabilized with Pluronic F-127-COOH surfactant;23 see Table 3) was rich in carboxylic groups prior to its decoration with the targeting residues. Importance of Quantification of the Decoration Efficiency. The presence of dozens of thousands of targeting residues on the surface of a single nano-DDS in our formulation (see Figure 4B) indicates high efficiency of the conjugation reactions. However, implications of these findings

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AUTHOR INFORMATION

Corresponding Author

*Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva 84105, Israel. Tel: +972-86477381. Fax: +972-8-6479303. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Breunig, M.; Bauer, S.; Goepferich, A. Polymers and nanoparticles: intelligent tools for intracellular targeting? Eur. J. Pharm. Biopharm 2008, 68, 112−28. (2) D’Souza, G. G.; Weissig, V. Subcellular targeting: a new frontier for drug-loaded pharmaceutical nanocarriers and the concept of the magic bullet. Expert Opin. Drug Delivery 2009, 6, 1135−48.

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(3) Stepensky, D. Quantitative aspects of intracellularly-targeted drug delivery. Pharm. Res. 2010, 27, 2776−80. (4) Yu, M. K.; Park, J.; Jon, S. Targeting strategies for multifunctional nanoparticles in cancer imaging and therapy. Theranostics 2012, 2, 3− 44. (5) Sneh-Edri, H.; Likhtenshtein, D.; Stepensky, D. Intracellular targeting of PLGA nanoparticles encapsulating antigenic peptide to the endoplasmic reticulum of dendritic cells and its effect on antigen crosspresentation in vitro. Mol. Pharmaceutics 2011, 8, 1266−75. (6) Joshi, V. B.; Geary, S. M.; Salem, A. K. Production of antigenloaded biodegradable nanoparticles and uptake by dendritic cells. Methods Mol. Biol. 2014, 1139, 453−66. (7) Vasir, J. K.; Labhasetwar, V. Preparation of Biodegradable Nanoparticles and Their Use in Transfection. Cold Spring Harbor Protoc. 2008, DOI: 10.1101/pdb.prot4888. (8) Jiang, X.; Zhang, J.; Zhou, Y.; Xu, J.; Liu, S. Facile preparation of core-crosslinked micelles from azide-containing thermoresponsive double hydrophilic diblock copolymer via Click chemistry. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 860−71. (9) Lallana, E.; Sousa-Herves, A.; Fernandez-Trillo, F.; Riguera, R.; Fernandez-Megia, E. Click chemistry for drug delivery nanosystems. Pharm. Res. 2012, 29, 1−34. (10) Avti, P. K.; Maysinger, D.; Kakkar, A. Alkyne-azide “click” chemistry in designing nanocarriers for applications in biology. Molecules 2013, 18, 9531−49. (11) Alsante, K. M.; Huynh-Ba, K. C.; Baertschi, S. W.; Reed, R. A.; Landis, M. S.; Furness, S.; Olsen, B.; Mowery, M.; Russo, K.; Iser, R.; Stephenson, G. A.; Jansen, P. Recent trends in product development and regulatory issues on impurities in active pharmaceutical ingredient (API) and drug products. Part 2: Safety considerations of impurities in pharmaceutical products and surveying the impurity landscape. AAPS PharmSciTech 2014, 15, 237−51. (12) Brem, S.; Grossman, S. A.; Carson, K. A.; New, P.; Phuphanich, S.; Alavi, J. B.; Mikkelsen, T.; Fisher, J. D.; New Approaches to Brain Tumor Therapy CNS Consortium. Phase 2 trial of copper depletion and penicillamine as antiangiogenesis therapy of glioblastoma. NeuroOncology 2005, 7, 246−53. (13) Ishak, R.; Abbas, O. Penicillamine revisited: historic overview and review of the clinical uses and cutaneous adverse effects. Am. J. Clin. Dermatol. 2013, 14, 223−33. (14) Stern, B. R.; Solioz, M.; Krewski, D.; Aggett, P.; Aw, T. C.; Baker, S.; Crump, K.; Dourson, M.; Haber, L.; Hertzberg, R.; Keen, C.; Meek, B.; Rudenko, L.; Schoeny, R.; Slob, W.; Starr, T. Copper and human health: biochemistry, genetics, and strategies for modeling dose-response relationships. J. Toxicol. Environ. Health, Part B 2007, 10, 157−222. (15) Lanone, S.; Rogerieux, F.; Geys, J.; Dupont, A.; MaillotMarechal, E.; Boczkowski, J.; Lacroix, G.; Hoet, P. Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines. Part. Fibre Toxicol. 2009, 6, 14. (16) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793−7. (17) Jewett, J. C.; Bertozzi, C. R. Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 2010, 39, 1272−9. (18) Zhang, B.; Yan, B. Analytical strategies for characterizing the surface chemistry of nanoparticles. Anal. Bioanal. Chem. 2010, 396, 973−82. (19) Weingart, J.; Vabbilisetty, P.; Sun, X. L. Membrane mimetic surface functionalization of nanoparticles: methods and applications. Adv. Colloid Interface Sci. 2013, 197−198, 68−84. (20) Keegan, M. E.; Falcone, J. L.; Leung, T. C.; Saltzman, W. M. Biodegradable microspheres with enhanced capacity for covalently bound surface ligands. Macromolecules 2004, 37, 9779−84. (21) Keegan, M. E.; Royce, S. M.; Fahmy, T.; Saltzman, W. M. In vitro evaluation of biodegradable microspheres with surface-bound ligands. J. Controlled Release 2006, 110, 574−80.

(22) Danhier, F.; Ansorena, E.; Silva, J. M.; Coco, R.; Le Breton, A.; Preat, V. PLGA-based nanoparticles: an overview of biomedical applications. J. Controlled Release 2012, 161, 505−22. (23) Chittasupho, C.; Manikwar, P.; Krise, J. P.; Siahaan, T. J.; Berkland, C. cIBR effectively targets nanoparticles to LFA-1 on acute lymphoblastic T cells. Mol. Pharmaceutics 2010, 7, 146−55. (24) Ruenraroengsak, P.; Cook, J. M.; Florence, A. T. Nanosystem drug targeting: Facing up to complex realities. J. Controlled Release 2010, 141, 265−76. (25) Moghimi, S. M.; Hunter, A. C.; Andresen, T. L. Factors controlling nanoparticle pharmacokinetics: an integrated analysis and perspective. Annu. Rev. Pharmacol. Toxicol. 2012, 52, 481−503. (26) Kozlovskaya, L.; Stepensky, D. Quantitative analysis of the brain-targeted delivery of drugs and model compounds using nanodelivery systems. J. Controlled Release 2013, 171, 17−23. (27) Park, J.; Mattessich, T.; Jay, S. M.; Agawu, A.; Saltzman, W. M.; Fahmy, T. M. Enhancement of surface ligand display on PLGA nanoparticles with amphiphilic ligand conjugates. J. Controlled Release 2011, 156, 109−15. (28) Zhao, J.; Mi, Y.; Liu, Y.; Feng, S. S. Quantitative control of targeting effect of anticancer drugs formulated by ligand-conjugated nanoparticles of biodegradable copolymer blend. Biomaterials 2012, 33, 1948−58. (29) Fakhari, A.; Baoum, A.; Siahaan, T. J.; Le, K. B.; Berkland, C. Controlling ligand surface density optimizes nanoparticle binding to ICAM-1. J. Pharm. Sci. 2011, 100, 1045−56. (30) Elias, D. R.; Poloukhtine, A.; Popik, V.; Tsourkas, A. Effect of ligand density, receptor density, and nanoparticle size on cell targeting. Nanomedicine 2013, 9, 194−201. (31) Hoshino, A.; Fujioka, K.; Oku, T.; Nakamura, S.; Suga, M.; Yamaguchi, Y.; Suzuki, K.; Yasuhara, M.; Yamamoto, K. Quantum dots targeted to the assigned organelle in living cells. Microbiol. Immunol. 2004, 48, 985−94. (32) Zhang, N.; Chittasupho, C.; Duangrat, C.; Siahaan, T. J.; Berkland, C. PLGA nanoparticle–peptide conjugate effectively targets intercellular cell-adhesion molecule-1. Bioconjugate Chem. 2008, 19, 145−52. (33) Lu, J.; Shi, M.; Shoichet, M. S. Click chemistry functionalized polymeric nanoparticles target corneal epithelial cells through RGDcell surface receptors. Bioconjugate Chem. 2009, 20, 87−94. (34) Wang, Z.; Chui, W. K.; Ho, P. C. Design of a multifunctional PLGA nanoparticulate drug delivery system: evaluation of its physicochemical properties and anticancer activity to malignant cancer cells. Pharm. Res. 2009, 26, 1162−71. (35) Toti, U. S.; Guru, B. R.; Grill, A. E.; Panyam, J. Interfacial activity assisted surface functionalization: a novel approach to incorporate maleimide functional groups and cRGD peptide on polymeric nanoparticles for targeted drug delivery. Mol. Pharmaceutics 2010, 7, 1108−17. (36) Misra, R.; Sahoo, S. K. Intracellular trafficking of nuclear localization signal conjugated nanoparticles for cancer therapy. Eur. J. Pharm. Sci. 2010, 39, 152−63.

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