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Enhanced Drug Delivery into Cell Cytosol via Glycoprotein H‑Derived Peptide Conjugated Nanoemulsions Teresa Fotticchia,†,⊥ Raffaele Vecchione,*,†,‡,⊥ Pasqualina Liana Scognamiglio,† Daniela Guarnieri,+ Vincenzo Calcagno,† Concetta Di Natale,† Chiara Attanasio,† Maria De Gregorio,† Chiara Di Cicco,† Vincenzo Quagliariello,§ Nicola Maurea,∥ Antonio Barbieri,# Claudio Arra,# Luca Raiola,† Rosario V. Iaffaioli,§ and Paolo A. Netti†,‡ †
Center for Advanced Biomaterials for Health Care@CRIB, Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, Napoli 80125, Italy ‡ Interdisciplinary Research Center of Biomaterials, CRIB, University Federico II, P.le Tecchio 80, Naples 80125, Italy + Nanobiointeractions & Nanodiagnostics, Istituto Italiano di Tecnologia (IIT), Via Morego, 30, Genova 16163, Italy § Medical Oncology, Abdominal Department, ∥Division of Cardiology, and #Animal Facility Unit, Department of Research, National Cancer Institute G. Pascale Foundation, Napoli 80131, Italy S Supporting Information *
ABSTRACT: The key role of nanocarriers in improving the pharmacological properties of commonly used drugs is recognized worldwide. It is also known that in the development of new effective nanocarriers the use of targeting moieties integrated on their surface is essential. Herein, we propose a nanocarrier based on an oil in water nanoemulsion coated with a membranotropic peptide derived from the glycoprotein H of Herpes simplex virus 1, known as gH625, in order to reduce endolysosomal accumulation and to enhance cytosolic localization. In addition, we show an enhanced anti-inflammatory activity of curcumin, a bioactive compound isolated from the Curcuma longa plant, when loaded into our engineered nanocarriers. This effect is a consequence of a higher uptake combined with a high curcumin preservation exerted by the active nanocapsules compared to control ones. When loaded into our nanocapsules, indeed, curcumin molecules are directly internalized into the cytosol rather than into lysosomes. Further, in order to extend the in vitro experimental setting with a more complex model and to explore the possibility to use our nanocarriers for further biological applications, we tested their performance in a 3D sprouting angiogenesis model. Finally, we show promising preliminary in vivo results by assessing the anti-inflammatory properties of the proposed nanocarrier. KEYWORDS: nanoemulsions, nanocapsules, curcumin, gH625, drug delivery under preclinical and clinical stage of development.15 Further, it was shown that O/W NEs are able to actively target cells or tissues when specific moieties coat their surface.7 In this work, we developed O/W NEs which mostly accumulate into cytosol rather than into lysosomes thanks to the conjugation to a membranotropic peptide through supramolecular nanoassembly. This 19-residues peptide, identified as gH625, is derived from the glycoprotein H (gH) of Herpes simplex virus 1 and belongs to a class of hydrophobic peptides able to induce a
O
il in water nanoemulsions (O/W NEs) are an ideal vehicle for drug delivery thanks to their ability to dissolve large quantities of hydrophobic drugs as well as contrasting agents.1−6 There is currently a great deal of research in this field, especially because of the poor water solubility of many newly discovered drugs,7 including natural drugs such as nutraceuticals which are mainly lipophilic.8−11 In addition, O/W NEs are able to protect drugs from hydrolysis and enzymatic degradation under physiological conditions and to be properly stabilized as previously reported.8,12−14 These basic properties make O/W NEs promising drug delivery systems, and a number of pharmaceutical formulations based on them are already on the market, while several others are © 2017 American Chemical Society
Received: May 3, 2017 Accepted: August 18, 2017 Published: August 18, 2017 9802
DOI: 10.1021/acsnano.7b03058 ACS Nano 2017, 11, 9802−9813
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Cite This: ACS Nano 2017, 11, 9802-9813
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ACS Nano temporary and local membrane destabilization with following reorganization.16−18 This is the consequence of an efficient interaction between the peptide and the biological membranes that promotes lipid membrane-reorganizing processes, such as fusion or pore formation.17−19 We recently demonstrated that surface decoration of rigid polymer nanoparticles with gH625 enables their entrance into the cells through a nonconventional path and their crossing through a confluent layer of mouse microvascular endothelial cells (bEnd.3) both in static and in dynamic conditions.19−21 In the case of metallic platinum ultrasmall nanoparticles, it partially increases their cytosolic delivery and anti-oxidant properties in the HeLa cell line.22 In this work we show that although the described extra-lysosomal path is a peculiar mechanism of entrance of nanoparticles into the cells, it can easily be translated to soft matter nanocarriers such as O/W NEs. Herein, a ternary poly-L-lysine-streptavidingH625 supramolecular structure was assembled for O/W NEs decoration. To this end, we first functionalized the gH625 sequence with a biotinylated poly(ethylene glycol) (PEG) chain in order to inhibit the nanocarriers clearance by eluding the mononuclear phagocyte system (MPS), as highly reported among PEG properties.23 Then, we stabilized O/W NEs with a thin layer of biotinylated poly-L-lysine, and, after the addition of streptavidin, the biotin-PEG-gH625 molecule was deposited by an additive easy approach that exploits biotin−streptavidin physical interaction. Isothermal titration calorimetry (ITC) was used to evaluate the right amount of each component to be added following a step by step procedure. Afterward, these nanocarriers were characterized in terms of their chemical and morphological features. Then, a biological study was performed in order to assess the effectiveness of the alternative penetration pathway of the proposed O/W NEs based on enhanced cytosolic localization. To this aim, we compared the performance of O/W NE functionalized with gH625 to that of the same system conjugated with a nonactive peptide in a monolayer of bEnd.3 cells. Subsequently, we confirmed the relevance of this result by loading both the systems with curcumin and comparing their anti-inflammatory activity in primary isolated cells, namely human umbilical vein endothelial cells (HUVEC). This test demonstrated that not only the anticancer activity of curcumin is higher when this molecule is delivered directly into the cytosol24 but also its anti-inflammatory effect is enhanced. Finally, in order to explore the ability of our O/W NEs to properly interact with cells in a 3D setting, we performed a sprouting angiogenesis assay based on the use of HUVEC spheroids. Then, we investigated the anti-inflammatory properties of the proposed nanocarrier in a mouse model of inflammation, confirming in vivo the effectiveness of our system.
Figure 1. Schematic representation of gH625-NCs multistep assembly. In the first step, the nanoemulsion is coated with PLLbiotin, and then in the second step, streptavidin is conjugated to the PLL-biotin-based SNEs. In the third step, the previously synthesized block of biotinylated PEG-peptide (active 3a and scramble 3b) is added to the preformed NCs acting streptavidin as a linker between PLL-biotin-based SNEs and the biotinylated PEGpeptide.
terminal peptide gH625 is able to cross the membrane bilayer, thus allowing the transport of the NCs into the cytoplasm. The functionalization of poly-L-lysine with biotin was detected by nuclear magnetic resonance (NMR) (Figure 2A). The peak at 3.0 ppm was assigned to the epsilon protons of free Lys (as confirmed by the spectrum of free poly-L-lysine shown in Figure 2B), whereas the peak at 2.9 ppm was assigned to the epsilon protons of Lys bound to biotin. The reaction scheme is reported in Figure 2C. The integration of those two peaks gives the percentage of biotin bound to poly-L-lysine in the PLLbiotin samples, showing a yield of functionalization of 5.0%. This degree of functionalization did not affect significantly the solubility of the modified polymer and at the same time guaranteed a PEG-peptide % lower than 10 as needed to let explicate PEG antiopsonic properties.27 The biotinylated SNE (1 wt % in oil and 0.0125 wt % in PLL-biotin) was prepared and characterized by dynamic light scattering (DLS) analysis which showed good results in terms of size, polydispersity index (PDI), and ζ-potential. In fact, we observed an increased size (154.3 nm) of the biotinylated SNEs as compared to the NE (137.3 nm) (Figure 3A) in combination with an inversion of charge, as expected. Indeed, the ζ-potential value was negative in the NE due to the presence of charged carboxylic acid groups along the lecithin chain (used as surfactant). Consequently, it became positive in the biotinylated SNEs due to the coating with the positively charged polymer (poly-L-lysine) (Figure 3B). In addition, DLS periodical measurements showed that the biotinylated SNE maintains its hydrodynamic diameter during the 6 weeks of investigation, thus demonstrating its good stability (Figure 3C). In order to establish the precise amount of streptavidin able to saturate all biotin groups exposed on the biotinylated SNE surface, we carried out ITC experiments. In this case, biotinylated SNE (at 0.5 wt % of oil), present in the cell, was titrated with a solution of streptavidin (10.41 μM) (Figure 4A). The ITC binding curve obtained from the integration of the heat data had a sigmoidal behavior and was fitted with an independent and equivalent-sites model. The best-fit parameters are reported in Table 1. As shown in Table 1, the affinity
RESULTS AND DISCUSSION Nanocapsule Preparation and Characterization. O/W NEs and poly-L-lysine-based secondary nanoemulsion (SNE) systems used here were previously reported and well characterized.13,25 To fabricate our systems, we used biotinylated poly-L-lysine (PLL-biotin), then the streptavidin is added and used as a linker between the PLL-biotin and the biotinylated PEG-peptide. In Figure 1 it is shown a schematic illustration of the decorated NCs assembly. The streptavidin bridges PLL-biotin and biotin-PEG-gH625 exploiting the streptavidin-biotin affinity. PEG is added to avoid NCs clearance by mononuclear phagocytic system in order to enhance their persistence in the blood.26,27 Moreover, the 9803
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Figure 2. 1H 1D NMR spectra of (A) biotin-PLL and (B) PLL in 90/10 H2O/D2O. (C) Schematic representation of poly-L-lysine functionalization with biotin.
between biotinylated SNE and streptavidin has very high Kd (Kd = 1/Kb) with values in the order of nM, as expected for a biotin−streptavidin interaction.28 The thermodynamic signature, traced by the thermodynamic parameters collected in Table 1 and depicted in Figure 4B, clearly shows that the interaction is exothermic and enthalpically driven. The latter condition is counterbalanced by an unfavorable entropic contribution to Gibbs energy. The favorable enthalpy is ascribable to the formation of new interactions between biotin and streptavidin upon binding. The unfavorable entropy contribution upon binding suggests that the critical conformational rearrangements due to the loss of conformational degrees of freedom for both the interacting molecules are predominant. To confirm that the obtained binding curve is only due to the interaction between biotin and streptavidin, the same experiment was performed without biotin. In particular, the same SNE was titrated with a solution of streptavidin (10.41 μM). Raw data (Figure 4C) showed only small peaks of dilution demonstrating that streptavidin and SNE did not interact. According to the NMR data, biotinylated SNE (0.5 wt % of oil and 0.00625 wt %) is 14.5 μM concentrated in biotin (corresponding to 5% of the total amount of NH2 of poly-Llysine). In addition, ITC data show that the same SNE saturated with streptavidin is 1.45 μM concentrated. This means that only 10% of biotin molecules are exposed on the SNE surface and able to bind the streptavidin added to the solution. ITC experiments allowed to calculate the exact amount of biotin exposed on the NC surface with respect to the
total quantity. These data were of basic importance to move forward the decoration of the NCs by molecular assembly in order to avoid nonspecific interactions due to the addition of an amount of biomolecules that exceeds the available sites. Regarding the peptide synthesis, three glycines and a cysteine (as Fmoc-Cys(Mmt)-OH) were added to the C-terminal (gH625-GGGC). The three glycines acted as spacers in order to retain the innate peptide conformation when conjugated to the particle surface. FITC was added at the N-terminal spaced with a β-alanine in order to label the peptide sequence and to detect its conjugation with the NCs (FITC-gH625-GGGC). The PEGylated peptide was produced by selectively removing the Mmt protector group from the thiol group of the last cysteine residue, when the peptide was still linked to the resin. Then, the maleimide group of a bifunctionalized polyethylenglycol (Mal-PEG-biotin, 2000 Da) was coupled obtaining the biotin-PEGylated peptide (FITC-gH625-PEG-biotin). PEG 2000 was chosen for its well-known capability to extend the circulation time of the NCs and to decrease its accumulation into the liver.29 In literature it is reported that PEG 2000nanoemulsion had a 7-fold increased half-life after injection in rats.30 The schematic representation of solid-phase peptide synthesis and the characterization by high-pressure liquid chromatography are reported in the Supporting Information (Figure S1). The secondary structure of gH625-GGGC and gH625-GGGC-PEG was determined in water solution by CD spectroscopy in the far-UV spectral region (195−260 nm). CD spectra of gH625-GGGC revealed the presence of a random 9804
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Figure 3. (A) Size data of O/W NEs (black) compared with biotinylated SNEs (red). (B) ζ-Potential data of NEs (black) compared with biotinylated SNEs (red). (C) Dimensional behavior over time for biotinylated SNEs measured by DLS analysis. Figure 4. (A) ITC data for titration by stepwise injections of streptavidin into biotinylated SNE (0.5 wt % of oil) at 25 °C. (B) Normalized heat of interaction between biotinylated SNE and streptavidin. The solid squares are the experimental data obtained through the integration of the raw data and subtracting the heat of ligand dilution into the buffer. The lines represent the best fit obtained with the independent-sites model. (C) ITC data for titration by stepwise injections of streptavidin into nonbiotinylated SNE (0.5 wt % of oil) at 25 °C.
coil conformation. This spectral characteristic is compliant with the CD spectra of gH625 reported in literature31 demonstrating that the insertion of amino acids at the C-terminal does not alter peptide conformation. Further, the gH625-GGGC-PEG CD spectra showed the same trend proving that the long PEG chain does not interfere with the peptide secondary structures (Figure 5). Pure FITC-gH625-PEG-biotin was then added to the streptavidin-biotinylated-SNE under sonication at a 1:1 streptavidin-biotin molar ratio. The completely functionalized NCs were finally characterized by DLS (Figure 6). Their hydrodynamic diameter was ∼156 nm, as illustrated in Table 2. In agreement with the formation of the coating, fluorescent NCs were detected by confocal microscopy (Figure S2). In the same way, pure FITC-CTRLgH625, acting as a negative control peptide, was synthesized on solid phase, PEGylated, and conjugated to the streptavidin-biotinylatedSNE surface with the same procedure used for gH625. All of the properties, in terms of size, PDI, and ζ-potential, of all the nanocarriers are reported in Table 2. In Vitro Experiments. To study the effect of the conjugation of the gH625 to the NC surface, we performed uptake experiments, and the results were compared with those obtained using CTRLgH625-NCs. The bEnd.3 cells are typically used as a blood brain barrier in vitro 2D model due to their rapid growth end-capability to form a confluent monolayer that mimics the endothelium.20,32 In order to assess the ability of gH625 to affect the mechanisms of O/W SNE internalization, we performed confocal microscope analyses to investigate the colocalization of NCs with lysosomes. Lysosomes were stained with lysotraker
Table 1. Thermodynamic Parameters of Interaction between Streptavidin and Biotinylated SNE at 25 °C molar enthalpy ΔH° (kJ mol−1)
entropic contribution TΔS° (kJ mol−1)
Gibbs energy ΔG° (25 °C) (kJ mol−1)
binding constant Kb (M−1)
−510.1
−463.24
−46.86
1.6 × 108
(red), while NCs were stained with the FITC-conjugated peptide (green). After 24 h of incubation, it was shown that gH625-NCs penetrated into the cells and were localized outside the lysosomes (Figure 7A), whereas CTRLgH625-NCs were mainly colocalized with them (Figure 7B). These data clearly indicate that the peptide changed the mechanism of nanoemulsion uptake by inducing an alternative penetration pathway as previously demonstrated with solid polystyrene nanoparticles.20 Since these experiments were performed on NCs following the emission of the FITC-peptide, in order to confirm that the entire nanocarrier was able to penetrate into the cells we performed further uptake experiments labeling also the oil core with a red lipophilic dye (Nile Red). As shown in Figure 7C−D, the fluorescence of the FITC-conjugated peptide and the fluorescence of the oil containing Nile Red matched, indicating that the peptide remains bound to the SNE during 9805
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Figure 5. CD spectra of gH625-GGGC (black) and gH625-GGGCPEG (red) in water. The spectra were recorded using 0.2 cm path length cells.
Figure 7. Colocalization of (A) gH625-NCs and (B) CTRLgH625NCs with lysosomes in bEnd.3 cells after 24 h of incubation. Green: NPs; red: lysotracker. Uptake of (C) gH625-NCs and (D) CTRLgH625-NCs both loaded with Nile Red after 24 h incubation in bEnd.3 cells. Green: peptide; red: oil; light blue: nuclei.
Figure 6. Size data of streptavidin-biotinylated-SNEs (black) compared with gH625-NCs (red).
Table 2. Size and ζ-Potential Measurements of NCs during the Four Steps of Assembly
O/W NE biotinylated SNE streptavidinbiotinylated SNE gH625-NCs CTRLgH625-NCs
size (nm)
PDI
ζ-potential (mV)
137.3 ± 0.56 153.5 ± 0.88 153.8 ± 0.56
0.041 ± 0.003 0.054 ± 0.013 0.075 ± 0.0063
−43.3 ± 1.90 45.9 ± 0.91 42.9 ± 0.83
Figure 8. Uptake mechanism of gH625-NCs (green spheres) and CTRLgH625-NCs (red spheres) in bEnd.3 cells.
155.9 ± 2.00 154.8 ± 2.90
0.099 ± 0.01 0.068 ± 0.02
47.5 ± 0.60 48.6 ± 3.39
activity when localized in the cytosol rather than into lysosomes.24 It is also known that several neurological diseases including brain tumors are related to a disruption of the blood brain barrier induced by inflammation.33 Therefore, as an applicative example, we exposed HUVEC cells to LPS-induced inflammation. 34 We then measured their inflammatory response to inflammation when treated with gH625-NCs or CTRLgH625-NCs. Considering the well-established antiinflammatory activity of curcumin,35 we promoted IL-8 and IL-6 production by incubating the cells with LPS (40 ng/mL). HUVEC incubation with LPS increased significantly interleukins production through the stimulation of toll like receptor type 4 (TLR4). When pretreated with free curcumin, CTRLgH625-NCs or gH625-NCs both at 5 and 15 μM concentrations, the levels of IL-6 and IL-8 decreased significantly (Figure 9). In brief, the anti-inflammatory activities are summarized in Table 3, where the best performance of gH625-NCs loaded with curcumin compared to CTRLgH625NCs is evident.
the uptake. Therefore, we are showing that soft matter such as O/W SNEs can be internalized by bEnd.3 cells following the extra-lysosomal path if they are conjugated with the gH625 peptide. This peculiar feature may be critical to deliver high amounts of lipophilic drugs into the cells. This means that all drugs that would be potentially damaged by the lysosomal environment may be delivered following the proposed approach. A schematic representation of the hypothesized cell uptake mechanism by means of the gH625-NCs and CTRLgH625NCs is reported in Figure 8. In order to support this hypothesis, we used a model molecule. We chose curcumin, a nutraceutical molecule, which intracellular activity is connected to its localization. In fact, it was demonstrated that curcumin exerts a higher anticancer 9806
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Figure 9. Anti-inflammatory properties of free curcumin, CTRLgH625-NCs, and gh625-NCs (both nanocarriers are loaded with the indicated concentrations of curcumin) evaluated by IL-8 and IL-6 production in HUVECs. Cells were treated for 5 h before being exposed to LPS (40 ng/mL) for 12 h.
linear trend over time. Interestingly, gH625-NCs showed always higher cellular uptake compared to CTRLgh625-NCS (p < 0.01), therefore indicating the key role of the peptide in nanocarrier uptake into human vascular endothelium cells. Intriguingly, the enhanced anti-inflammatory effect was due not only to the higher uptake but also to the different mechanism of internalization (Figure 10, lower panel). These results also add knowledge on the properties of curcumin which results to be more active when released into the cytosol. In order to explore further biological applications of our O/ W NCs, we tested them in a 3D in vitro model of angiogenesis. It is well-known that angiogenesis plays a key role in both physiological and pathological conditions such as cancer and chronic inflammation as well as tissue regeneration. Curcumin directly inhibits angiogenesis and downregulates the expression of proangiogenic factors by targeting several molecular pathways.36 In addition, it has been shown that nanoformulation of curcumin protects HUVEC cells from inflammation, atherosclerosis, and oxidative stress.37,38 In our sprouting angiogenesis assay we focused on a double effect: drug encapsulation and peptide conjugation. These features were expected to enhance curcumin uptake compared to free drug and to allow curcumin release into cell cytosol rather than into lysosomes. During cell seeding as well as during spheroid formation and culture, we exposed HUVECs to free curcumin, curcumin loaded monolayer conjugated with active peptide (CMgH625), and curcumin loaded monolayer conjugated with the CTRLgH625peptide. After 72 h, we observed a
Table 3. Anti-Inflammatory Activities for IL-6 and IL-8 for Free Curcumin, Active, and Control NCs
treatments
reduction of interleukin secretion compared to unpretreated cells (exposed only to LPS) (%), IL-8
Free Curcumin (μM) 5 9.3 15 25.2 CTRLgH625-NCs (μM) 5 11.4 15 34.3 gH625-NCs (μM) 5 40.5 15 66.5
p value
reduction of interleukin secretion compared to unpretreated cells (exposed only to LPS) (%), IL-6
p value
0.09 0.004
15.3 30.3
0.07 0.007
0.1 0.03
18.2 39.2
0.02 0.002
0.001 0.0001
42.0 65.6
0.002 0.0004
As clearly shown in Table 3, remarkably, the most effective anti-inflammatory results were obtained with the pretreatment with gH625-NCs that reduced the production of IL-8 and IL-6 of 40.5 and 42% at 5 μM (p = 0.001; 0.002, respectively) and of 67 and 66% at 15 μM of curcumin concentrations as compared to unpreteated cells (p = 0.001; 0.004, respectively). Therefore, we carried out an uptake quantification of the two nanocarriers, by labeling the oil core with FITC, in order to elucidate the reason for the higher anti-inflammatory activity of the gH625NCs as compared to the control ones. As clearly shown in Figure 10 (upper panel), the uptake of both fluorescent nanocarriers has a time-dependent value in HUVEC cells with a 9807
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Figure 10. Upper panel: Fluorescent CTRLgH625-NCS and gH625-NCS uptake quantification by HUVEC cells from 0.5 up to 24 h of treatment. Lower panel: Anti-inflammatory activity expressed as percentage of IL-6 and IL-8 production inhibition and cellular uptake expressed as percentage of relative uptake after 6 h of incubation of HUVEC cells with the two systems.
to ∼24 and 35% IL-6 and IL1-β skin production compared to those functionalized with the scramble peptide (CTRLgH625NCs) (p = 0.05 and 0.01, respectively) indicating a more efficient cellular internalization of the gH625-NCs also in vivo. Considering the crucial role of some interleukins and cytokines in the genesis of cardio-vascular diseases,40,41 it could be interesting to consider the use of such a nanocarrier loaded with curcumin as an anti-inflammatory system to prevent vascular diseases related to inflammation, ventricular remodeling, and exercise capacity.
proangiogenic effect of curcumin when it was encapsulated in gH625-NCS. In fact, the average sprout number and confocal images of treated spheroids reported in Figure 11 show that the group that received the free drug displayed a significant antiangiogenic effect compared to the positive control, corresponding to a 57% decrease in average sprout number, a value almost equal to the negative control. In contrast, curcumin loaded NCs compared to the positive control showed a proangiogenic effect of 21% and 35% when conjugated with the CTRLgH625 and the functional gH625 peptide, respectively. These findings corroborate the hypothesis that the conjugation of gH625 peptide to drug loaded O/W NEs modulates the effect of the encapsulated molecule. In Vivo Experiments. Finally, a preliminary in vivo test has been carried out as a more realistic system validation. As shown in Figure 12, treatment with LPS increased IL-6 and IL1-β skin production to ∼47.5 and 28.6% compared to PBS treated mice (p < 0.001 in both cases). This behavior is probably related to the stimulation of TLR4 by LPS. These receptors are expressed on the membrane of several skin cells and lead to an upregulation of interleukin expression and secretion.35,39 Subcutaneous administration of unformulated curcumin (Free Curc) slightly decreased inflammatory cytokine production to ∼11 and 19% for IL-6 and IL1-β, respectively. When encapsulated, curcumin anti-inflammatory activity was enhanced providing the best results in the case of the active peptide (gH625-NCs), which reduced to 50 and 70% IL-6 and IL1-β skin production, respectively, in comparison to LPS treated mice (p < 0.001 in both cases). Nanocarriers functionalized with the active peptide (gH625-NCs) reduced
CONCLUSIONS In this work, we present nanoemulsions effectively functionalized with a peptide, namely gH625, capable of merging with biological membranes. In particular, the peptide is conjugated to a biotinylated PEG chain in order to exploit both the antifouling properties of the polymer and the strong physical interaction between biotin and streptavidin. Indeed, nanoemulsions are first coated with a biotin-poly-L-lysine, while streptavidin is used as linker between the two biotinylated components of the nanocarrier. Due to the softness of the nanocarrier, we exploited a mild additive approach based on biotin−streptavidin bonding which does not involve any chemical reaction for the preparation of the nanocarrier and, therefore, does not require any purification step. For assembly optimization, an isothermal titration calorimetry (ITC) experiment was carried out to evaluate the right amount of components to be added step by step. The capability of gH625 peptide to condition the entrance of rigid polymeric nanoparticles when used to decorate their surface has been 9808
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Figure 11. Upper panel: Quantitative analysis of the proangiogenic activity of curcumin loaded O/W NC conjugated with scramble (CTRLgH625-NCs) or active peptide (gH625-NCs) compared to free curcumin and CTRLs. HUVEC spheroids were exposed for 72 h to the different curcumin formulations (curcumin concentration 15 μM). The graph displays a proangiogenic effect of encapsulated curcumin that is significantly stronger in the gH625-NCs group. In contrast, the group treated with the free drug shows the same reactivity of the CTRL-group. p values
