Oligoarginine Peptide Conjugated to BSA Improves Cell Penetration of Gold Nanorods and Nanoprisms for Biomedical Applications

Gold nanoparticles (AuNPs) have been shown to be outstanding tools for drug delivery and biomedical applications, mainly owing to their colloidal stability, surface chemistry, and photothermal properties. The biocompatibility and stability of nanoparticles can be improved by capping the nanoparticles with endogenous proteins, such as albumin. Notably, protein coating of nanoparticles can interfere with and decrease their cell penetration. Therefore, in the present study, we functionalized albumin with the r8 peptide (All-D, octaarginine) and used it for coating NIR-plasmonic anisotropic gold nanoparticles. Gold nanoprisms (AuNPrs) and gold nanorods (AuNRs) were coated with bovine serum albumin (BSA) previously functionalized using a cell penetrating peptide (CPP) with the r8 sequence (BSA-r8). The effect of the coated and r8-functionalized AuNPs on HeLa cell viability was assessed by the MTS assay, showing a low effect on cell viability after BSA coating. Moreover, the internalization of the nanostructures into HeLa cells was assessed by confocal microscopy and transmission electron microscopy (TEM). As a result, both nanoconstructs showed an improved internalization level after being capped with BSA-r8, in contrast to the BSA-functionalized control, suggesting the predominant role of CPP functionalization in cell internalization. Thus, our results validate both novel nanoconstructs as potential candidates to be coated by endogenous proteins and functionalized with a CPP to optimize cell internalization. In a further approach, coating AuNPs with CPP-functionalized BSA can broaden the possibilities for biomedical applications by combining their optical properties, biocompatibility, and cell-penetration abilities.


AuNRs Synthesis
AuNRs were synthesized using a previously reported seed-mediated procedure [64]. Briefly, 5 mL of a 0.3 mM HAuCl 4 solution in CTAB 0.1 M was reduced by ice-cold NaBH 4 10 mM (300 µL), resulting in a brownish-yellow seed solution. Then, 10 mL of a 0.5 mM HAuCl 4 solution (in CTAB 0.1 M) was reduced by ascorbic acid in the presence of AgNO 3 , until it reached a colorless growth solution. Finally, 120 µL of the seed solution was added to the growth solution and allowed to rest for 30 min in a thermostatic bath at 27 • C. The obtained AuNRs were centrifuged at 7030 g for 30 min, and the pellet was resuspended in Milli-Q water.

AuNPrs Synthesis
AuNPrs were obtained by Na 2 S 2 O 3 reduction of HAuCl 4 , as previously reported [28,36]. A 2 mM HAuCl 4 solution was first reduced by 0.6 mM Na 2 S 2 O 3 and allowed to rest for 9 min. Then, a second addition of 0.6 mM Na 2 S 2 O 3 was performed, and the solution was left undisturbed for 30 min. The purple solution containing the AuNPs was centrifuged and resuspended in Milli-Q water. PEG functionalization was achieved at pH = 12 by adding 15 µL of a 2.7 mM HS-PEG5000-COOH solution and allowing conjugation under a magnetic stirrer for 3 h. Finally, AuNPrs were separated from smaller undesired AuNPs using a successive differential centrifugation procedure, as reported [36].

Characterization of the Nanoparticles
AuNRs and AuNPrs were characterized before and after BSA-r 8 coating by UV-Vis-NIR absorption spectra, using a Lambda 25 spectrophotometer (Perkin Elmer, Waltham, MA, USA). Dynamic light scattering (DLS) and Z potential measurements were acquired in PBS pH = 7, at 25 • C, using a Zetasizer 3000 (Malvern Instruments, Malvern, UK), as triplicates in aqueous solution at 25 • C.
TEM images of AuNRs were acquired with a JEOL JEM-1010 microscope (JEOL USA, Peabody, MA, USA), using Formvar carbon-coated copper microgrids (200 mesh; Ted Pella, Redding, CA, USA). For AuNPrs, TEM images were obtained using a Philips CM 120 transmission electron microscope with an accelerating voltage of 120 kV and a 300 mesh Formvar/Carbon-Coated Copper grid. For both AuNPs, liquid suspensions were deposited on the microgrid and allowed to stand overnight before TEM image acquisition.

Peptide Synthesis
We synthesized the all-D peptide derived from D-amino acids for this study due to their enhanced enzymatic stability [65]. Arginine-rich peptide (r 8 ) was synthesized by solid phase peptide synthesis (SPPS), using an H-Rink Amide Protide resin (loading: 0.56 mmol/g) in a Liberty Blue™ Automated Microwave Peptide Synthesizer. Linear D-OctoArginine was synthesized on a 0.5 mmol scale using a 5 excess of Fmoc-amino acid (0.2 M), relative to the resin. The Bromo acetic acid was coupled using 2 cycles of 30 min of 4 equivalents of OxymaPure, followed by 4 equivalents of N,N -Diisopropylcarbodiimide, and then 4 equivalents of bromo acetic acid. Fmoc deprotection was carried out using 10% (w/v) piperazine and 0.1 M OxymaPure in a 9:1 mixture of NMP and EtOH. The  The peptide was purified by  semi-preparative HPLC on a Waters 2700 sample manager, equipped with a Waters 2487  dual-wavelength absorbance detector, a Waters 600 controller, a Waters fraction collector and Masslynx software by using a Sunfire C18 column (150 × 10 mm × 3.5 µm, 100 Å, Waters), flow rate 6.6 mL/min; solvent A = 0.1% TFA in water and solvent B = 0.1% TFA in acetonitrile. Purity and identity were assessed by UPCL (Waters Acquity equipped with Acquity photodiode array detector, flux rate 0.610 mL/min, Acquity UPLC BEH C18 Column, 130 Å, 1.7 µm, 2.1 mm × 100 mm; solvents A = 0.045% TFA in water, and B = 0.036% TFA in acetonitrile) and UPLC-MS (Waters Acquity UPLC System equipped with ESI-SQ Detector2, flux rate 0.610 mL/min, Acquity UPLC BEH C18 Column, 130 Å, 1.7 µm, 2.1 mm × 100 mm; solvents A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile).
The crude compound was purified by RP-HPLC at a semi-preparative scale and characterized by UPLC and UPLC-MS spectrometry (Supplementary Section 1, Figures S1 and S2) to confirm the identity of the synthesized compound, obtaining high purity (>95%). Amino acid content was analyzed by amino acid analysis; results are presented in Supplementary Section 2, Table S1.

BSA Functionalization
BSA was functionalized with r 8 , as represented in Scheme 1, to improve the internalization properties of the nanoconstructs, and Atto-565-NHS-ester was used as a fluorescent probe for detection by fluorescence and confocal microscopy. solid phase peptide synthesis (SPPS), using an H-Rink Amide Protide resin (loading: 0.56 mmol/g) in a Liberty Blue™ Automated Microwave Peptide Synthesizer. Linear D-OctoArginine was synthesized on a 0.5 mmol scale using a 5 excess of Fmoc-amino acid (0.2 M), relative to the resin. The Bromo acetic acid was coupled using 2 cycles of 30 min of 4 equivalents of OxymaPure, followed by 4 equivalents of N,N′-Diisopropylcarbodiimide, and then 4 equivalents of bromo acetic acid. Fmoc deprotection was carried out using 10% (w/v) piperazine and 0.1 M OxymaPure in a 9:1 mixture of NMP and EtOH. The resin was cleaved using TFA/H2O/TIS (95%/2.5%/2.5%) for 6 h; the TFA was evaporated, dissolved in a 50/50 H2O/ACN solution, and lyophilized. The peptide was purified by semi-preparative HPLC on a Waters 2700 sample manager, equipped with a Waters 2487 dual-wavelength absorbance detector, a Waters 600 controller, a Waters fraction collector and Masslynx software by using a Sunfire C18 column (150 × 10 mm × 3.5 μm, 100 Å, Waters), flow rate 6.6 mL/min; solvent A = 0.1% TFA in water and solvent B = 0.1% TFA in acetonitrile. Purity and identity were assessed by UPCL (Waters Acquity equipped with Acquity photodiode array detector, flux rate 0.610 mL/min, Acquity UPLC BEH C18 Column, 130 Å , 1.7 µ m, 2.1 mm × 100 mm; solvents A = 0.045% TFA in water, and B = 0.036% TFA in acetonitrile) and UPLC-MS (Waters Acquity UPLC System equipped with ESI-SQ Detector2, flux rate 0.610 mL/min, Acquity UPLC BEH C18 Column, 130 Å , 1.7 µ m, 2.1 mm × 100 mm; solvents A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile).
The crude compound was purified by RP-HPLC at a semi-preparative scale and characterized by UPLC and UPLC-MS spectrometry (Supplementary Section 1, Figure S1 and S2) to confirm the identity of the synthesized compound, obtaining high purity (>95%). Amino acid content was analyzed by amino acid analysis; results are presented in Supplementary Section 2, Table S1.

BSA Functionalization
BSA was functionalized with r8, as represented in Scheme 1, to improve the internalization properties of the nanoconstructs, and Atto-565-NHS-ester was used as a fluorescent probe for detection by fluorescence and confocal microscopy. Scheme 1. Synthetic approach for BSA functionalization by r8 peptide. First, 2-iminothiolane reacts with BSA amine groups in the surface of the protein, giving rise to available SH free groups, followed by r8-Br reaction with the free SH groups.


Second step: r8 peptide functionalization Scheme 1. Synthetic approach for BSA functionalization by r 8 peptide. First, 2-iminothiolane reacts with BSA amine groups in the surface of the protein, giving rise to available SH free groups, followed by r 8 -Br reaction with the free SH groups.

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Second step: r 8 peptide functionalization To the previously prepared protein, 52 µL of a 100 mg/mL Br-CH 2 -r 8 solution was added in 4 intervals every 10 min, and the reaction was left overnight at 4 • C (ratio 5:1 r 8 -Br:BSA). The product was purified with a P10 G25 desalting column and analyzed by UV-Vis-NIR spectroscopy and amino acid analysis to determine the amount of r 8 per BSA molecule.

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Third step: amino acid analysis of BSA-r 8 Amino acid analysis was performed following an acid hydrolysis procedure, adding 12 N HCl and a known concentration standard (Υ-aminobutyric acid, 0.1 mM), and allowed to react at 110 • C for 72 h. Amino acid quantification was carried out in an HPLC-PDA AccQ-Tag (C18; 4 µm; 3.9 × 15 mm). The amino acid content on the functionalized BSA resulted in a 2.4 r 8 /BSA ratio, as shown in Supplementary Section 2, Table S1. Fluorescent labeling of BSA was conducted as represented in Scheme 2. To 10 mg of BSA-r 8 of the previous step, 10.7 µL of a 10 mg/mL solution of Atto 565 NHS ester was added and allowed to react for 1 h. The final product (BSA-r 8 -A565) was purified with a P10 G25 desalting column and characterized by UV-Vis spectroscopy.
Amino acid analysis was performed following an acid hydrolysis procedure, adding 12 N HCl and a known concentration standard (ϒ-aminobutyric acid, 0.1 mM), and allowed to react at 110 °C for 72 h. Amino acid quantification was carried out in an HPLC-PDA AccQ-Tag (C18; 4 μm; 3.9 × 15 mm). The amino acid content on the functionalized BSA resulted in a 2.4 r8/BSA ratio, as shown in Supplementary Section 2, Table  S1.

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BSA-r8 fluorescent labeling Fluorescent labeling of BSA was conducted as represented in Scheme 2. To 10 mg of BSA-r8 of the previous step, 10.7 µ L of a 10 mg/mL solution of Atto 565 NHS ester was added and allowed to react for 1 h. The final product (BSA-r8-A565) was purified with a P10 G25 desalting column and characterized by UV-Vis spectroscopy.

Scheme 2.
Synthetic approach for BSA-r8 functionalization by Atto565-NHS ester in the NH2 free groups of BSA.

Circular Dichroism
The BSA and BSA-r8 1.5 × 10 −5 M samples were prepared in PBS 0.1× in the same conditions. The spectra were acquired at 20 °C, recorded from 200 to 250 nm in triplicate and condensed into a single spectrum to reduce noise at a 1 nm/s rate in a JASCO J-815 instrument (JASCO, Easton, MA, USA), using a 1 mm pathlength quartz-cuvette. The secondary structure content was calculated using the CDPro CONTIN 2DP (AUG 1982) (2DP-SW PACK) version from JASCO.
The molar ellipticity at wavelength λ ([θmrw]) was calculated as follows: where θλ is the observed ellipticity (degrees) at wavelength λ, d is the pathlength (cm), and c is the protein concentration (g/mL) [66]. According to UV-Vis spectroscopy measurements, the degree of labeling (DOL) was calculated as follows: and correction factor Atto 565 (CF Atto 565 ) = 0. 16 As a result, the DOL of BSA-r 8 A565 was 0.66 mol Atto/mol BSA.

Circular Dichroism
The BSA and BSA-r 8 1.5 × 10 −5 M samples were prepared in PBS 0.1× in the same conditions. The spectra were acquired at 20 • C, recorded from 200 to 250 nm in triplicate and condensed into a single spectrum to reduce noise at a 1 nm/s rate in a JASCO J-815 instrument (JASCO, Easton, MA, USA), using a 1 mm pathlength quartz-cuvette. The secondary structure content was calculated using the CDPro CONTIN 2DP (AUG 1982) (2DP-SW PACK) version from JASCO.
The molar ellipticity at wavelength λ ([θ mrw ]) was calculated as follows: where θ λ is the observed ellipticity (degrees) at wavelength λ, d is the pathlength (cm), and c is the protein concentration (g/mL) [66].
2.6. Capping of AuNPs with BSA or BSA-r 8 BSA-r 8 or BSA capping of the AuNPs was achieved by incubation, as previously reported [36]. Briefly, to 1 mL of AuNRs or AuNPrs at a 1 nM concentration, a solution of BSA or BSA-r 8 was added (final concentration of BSA = 1 mg/mL in 0.1× PBS), incubating for 2 h at 4 • C in low-binding 1.5 mL centrifuge tubes (Eppendorf ® , Hamburg, Germany), and the samples were centrifuged at 10,000 rpm for 10 min. The pellet containing the BSA or BSA-r 8 coated AuNPs was resuspended in 0.1× PBS. The protein content was quantified on the supernatants using a Micro BCA™ Protein Assay Kit from Thermo Scientific™ (Pierce, Rockford, IL, USA), according to the manufacturer's specifications, in triplicate.

Cell Viability MTS Assays
HeLa cells (1 × 10 4 cells/well) were seeded in TC pretreated 96-well plates and allowed to attach at 37 • C for 24 h. The treatments were added and allowed to incubate for 24 h. The medium was then replaced by a phenol red-free medium containing the MTS/PMS containing reagent, following the manufacturer's recommendations. The absorbance of the culture medium after incubation for 1 h was recorded at 490 nm using a microplate reader. Cell viability was calculated with respect to a non-treatment control (live control). Each treatment and control were made in quintuplicate, minimum n = 3. Statistical analysis was performed using GraphPad Prism V5.01.

Confocal Microscopy
HeLa cells (3 × 10 5 cells/plate) were seeded on collagen pre-coated MatTek glassbottom culture dishes (MatTek Corporation, Ashland, MA, USA) and attached for 48 h at standard culture conditions. The medium was then replaced with fresh medium, and the treatments were added considering 1 nM of each AuNP and incubated (1 or 24 h of treatment). The cells were subsequently washed 3 times with PBS, and phenol red-free medium containing the LIVE/DEAD ® Cell Imaging Kit from ThermoFisher was added (Molecular Probes Inc., Eugene, OR, USA). Fluorescence was detected on a Zeiss LSM 880 laser scanning microscope (Zeiss, Berlin, Germany) with Airyscan, equipped with a CO 2 and temperature-controlled environmental chamber. Hoechst was excited with an Ar laser at 405 nm, and emission was recorded at 458 nm; Atto-565 was excited by a laser at 405 nm, and emission was recorded at 458 nm. Images were processed using Image J 1.52 p software.

Internalization Evaluated by Transmission Electron Microscopy
In a Petri dish (90 × 15 mm), 1 × 10 6 HeLa cells were seeded and incubated at 37 • C until a minimum confluence of 80%. The cells were then washed, the medium was replaced with a treatment containing-medium (1 nM of BSA or BSA-r 8 coated AuNPs), and the cells were incubated for 24 h. Afterwards, the cells were washed and fixed with glutaraldehyde 2.5% in PBS 0.1×. The cells were scraped and centrifuged, and the obtained pellet was fixed with OsO 4 1% in PBS for 90 min and embedded in Epon resin. The resin was sliced into slices of 80 nm thickness, and the slices were placed in a Cu grid and stained with Reynold's reagent and uranyLess staining kit, before visualization. TEM images were acquired in a JEOL JEM-1010 microscope (JEOL Ltd., Tokyo, Japan).  Figure S1). Then, BSA was functionalized with r 8 (2.4 r 8 /BSA) as described in the experimental section. The degree of BSA functionalization was determined by amino acid analysis, resulting in 2.4 r 8 /BSA. To determine if the functionalization of the protein led to a change in BSA secondary structure, circular dichroism spectra were obtained. Figure 1 shows the CD spectrum of BSA and BSA-r 8 , indicating that they shared a similar profile, with a non-significant change from 39% and 36% alpha helix content before and after r 8 functionalization, respectively.

Results and Discussion
BSA-r 8 was functionalized with a fluorescent probe, namely A565, a known and commonly used red fluorescent probe, obtaining a 0.66 A565/BSA-r 8 degree of labeling, which resulted in BSA-A565-r 8 . In this study, we tested whether AuNPrs were a suitable alternative to more-traditional AuNRs; in this sense, AuNPrs were synthesized by simply reducing Au 3+ with Na 2 S 2 0 3 , thus avoiding the involvement of cytotoxic agents [28]. AuNRs and AuNPrs were synthesized with a characteristic morphology, as TEM images show in Supplementary Section 3 ( Figures S3 and S4), with a predominant aspect ratio of 3.5 for AuNRs and a 60 nm edge-length for AuNPrs. r8/BSA) as described in the experimental section. The degree of BSA functionalization was determined by amino acid analysis, resulting in 2.4 r8/BSA. To determine if the functionalization of the protein led to a change in BSA secondary structure, circular dichroism spectra were obtained. Figure 1 shows the CD spectrum of BSA and BSA-r8, indicating that they shared a similar profile, with a non-significant change from 39% and 36% alpha helix content before and after r8 functionalization, respectively. BSA-r8 was functionalized with a fluorescent probe, namely A565, a known and commonly used red fluorescent probe, obtaining a 0.66 A565/BSA-r8 degree of labeling, which resulted in BSA-A565-r8. In this study, we tested whether AuNPrs were a suitable alternative to more-traditional AuNRs; in this sense, AuNPrs were synthesized by simply reducing Au 3+ with Na2S203, thus avoiding the involvement of cytotoxic agents [28]. AuNRs and AuNPrs were synthesized with a characteristic morphology, as TEM images show in Supplementary Section 3 ( Figures S3 and S4), with a predominant aspect ratio of 3.5 for AuNRs and a 60 nm edge-length for AuNPrs.
It has been demonstrated by several spectroscopic techniques that BSA is spontaneously adsorbed in the surface of AuNPs via S-Au bonds between the free SH groups of BSA and the gold atoms in the AuNP surface [42,[67][68][69][70][71]. Therefore, both AuNRs and AuNPrs were coated with BSA and the functionalized BSA-A565-r8 by incubation, as summarized in Table 1 and Figures 2 and 3. Figures 2 and 3 show the UV-Vis-NIR spectra, size, and Z potential of the BSA and BSA-A565-r8 functionalized AuNPs in PBS (pH = 7). For AuNRs, a shoulder around 558 nm appeared after BSA-A565-r8 protein coating (Figure 2a), as well as an increment in the intensity of the first plasmon in AuNPrs (Figure 3a), confirming the presence of A565 and therefore, the functionalization of BSA on the AuNPs surface. In addition, the second plasmon of both AuNPs was shifted after the BSA and BSA-r8-A565 coating, attributed to the modification in the dielectric constant of the AuNPs [72] (Table 1 and Figures 2a and 3a), due to the presence of the protein on the AuNPs surface, as reported in previous studies [36,[73][74][75]. It has been demonstrated by several spectroscopic techniques that BSA is spontaneously adsorbed in the surface of AuNPs via S-Au bonds between the free SH groups of BSA and the gold atoms in the AuNP surface [42,[67][68][69][70][71]. Therefore, both AuNRs and AuNPrs were coated with BSA and the functionalized BSA-A565-r 8 by incubation, as summarized in Table 1 and Figures 2 and 3. Figures 2 and 3 show the UV-Vis-NIR spectra, size, and Z potential of the BSA and BSA-A565-r 8 functionalized AuNPs in PBS (pH = 7). For AuNRs, a shoulder around 558 nm appeared after BSA-A565-r 8 protein coating (Figure 2a), as well as an increment in the intensity of the first plasmon in AuNPrs (Figure 3a), confirming the presence of A565 and therefore, the functionalization of BSA on the AuNPs surface. In addition, the second plasmon of both AuNPs was shifted after the BSA and BSA-r8-A565 coating, attributed to the modification in the dielectric constant of the AuNPs [72] ( Table 1 and Figures 2a and 3a), due to the presence of the protein on the AuNPs surface, as reported in previous studies [36,[73][74][75].

Effect of AuNRs and AuNRs-BSA-A565-r 8 on Cell Viability
One of the main limitations of using AuNRs in biomedical applications is the cytotoxicity associated with the CTAB on the AuNP surface as the stabilizing agent in the synthesis procedure [30]. In order to overcome this issue, AuNRs can be coated with different materials, such as BSA [39,[79][80][81][82] to increase their biocompatibility. In this regard, Figure 4 shows that the freshly synthesized AuNRs drastically decreased the viability of Hela cells, whereas AuNRs-A565-BSA-r 8 did not cause a significant effect between the 0.005-2.5 nM range at 48 h. Additionally, flow cytometry showed no effect of 1 nM AuNRs-A565-BSA-r 8 after 24 h of administration (Supplementary Section 5, Figure S6). Similar results were found for BSA-coated AuNPs in previous studies; the BSA coating on AuNRs improved their biocompatibility properties [83][84][85], highlighting the potential of coated AuNRs for bioapplications.

Confocal Microscopy
In a first step, we tested the internalization ability of the AuNPs capped wi non-CPP functionalized BSA, as shown in Figure 6. The comparison between the BS

Confocal Microscopy
In a first step, we tested the internalization ability of the AuNPs capped with non-CPP functionalized BSA, as shown in Figure 6. The comparison between the BSA, AuNR-BSA, and AuNPr-BSA (labeled with A565) signals showed that BSA on the AuNP surface was not able to promote cell uptake under the studied conditions (

Confocal Microscopy
In a first step, we tested the internalization ability of the AuNPs capped with non-CPP functionalized BSA, as shown in Figure 6. The comparison between the BSA, AuNR-BSA, and AuNPr-BSA (labeled with A565) signals showed that BSA on the AuNP surface was not able to promote cell uptake under the studied conditions (  Internalization of BSA-r8 and the AuNPs capped with BSA-r8 (labeled with A565) was subsequently assessed by confocal microscopy. As Figure 7 shows, functionalization Internalization of BSA-r 8 and the AuNPs capped with BSA-r 8 (labeled with A565) was subsequently assessed by confocal microscopy. As Figure 7 shows, functionalization with r 8 increased the uptake of the protein, as well as that of the protein-coated AuNRs and AuNPrs in the red channel, using the same established conditions as in Figure  Although discussing the exact mechanism of internalization of the proposed nanoconstructs was not the objective of our study, previous reports have indicated that nanoconstructs conjugated with CPPs are internalized in vesicles inside cells via endocytic uptake [29,51,59,88], while arginine-rich CPPs can electrostatically bind with the cell membranes, promoting translocation into the cells [29,51,59,88].
To confirm that the internalization observed through confocal microscopy corresponded to the functionalized AuNPs, and to elucidate the intracellular location of the nanoconstructs in Hela cells, we performed a TEM study, as indicated in the next section.

TEM for Cell Internalization
The internalization of the BSA-A565-r 8 -coated AuNPs was assessed after 1 h and 24 h of incubation. Supplementary Section 6 ( Figures S7 and S8) shows internalization at 1 h, indicating that the nanoconstructs started interacting with the cell membranes at this time, to become ready for the internalization process. At 24 h, in contrast, internalization of both AuNRs-BSA-A565-r 8 and AuNPr-BSA-A565-r 8 was considerable and observed under the studied conditions, as shown in Figures 8 and 9.
with r8 increased the uptake of the protein, as well as that of the protein-coated AuNRs and AuNPrs in the red channel, using the same established conditions as in Figure 7 ([BSA] = 5 µ M, [AuNR-BSA-A565-r8] and [AuNPr-A565-r8] = 1 nM), suggesting the prevalent role of the r8 for cell internalization in both nanoconstructs, regardless of their surface charge. Although discussing the exact mechanism of internalization of the proposed nanoconstructs was not the objective of our study, previous reports have indicated that nanoconstructs conjugated with CPPs are internalized in vesicles inside cells via endocytic uptake [29,51,59,88], while arginine-rich CPPs can electrostatically bind with the cell membranes, promoting translocation into the cells [29,51,59,88].
To confirm that the internalization observed through confocal microscopy corresponded to the functionalized AuNPs, and to elucidate the intracellular location of the nanoconstructs in Hela cells, we performed a TEM study, as indicated in the next section.

TEM for Cell Internalization
The internalization of the BSA-A565-r8-coated AuNPs was assessed after 1 h and 24 h of incubation. Supplementary Section 6 ( Figures S7 and S8) shows internalization at 1 h, indicating that the nanoconstructs started interacting with the cell membranes at this time, to become ready for the internalization process. At 24 h, in contrast, internalization of both AuNRs-BSA-A565-r8 and AuNPr-BSA-A565-r8 was considerable and observed under the studied conditions, as shown in Figures 8 and 9.     The TEM images allowed determination of the precise location of the AuNPs in the cells. Both AuNRs and AuNPrs-based nanocarriers seemed to be located inside the cells and accumulated into vesicles in the cytoplasm, in agreement with the proposed endocytic uptake [29,51,59,88]. According to Liu et al. [89], the vesicles were labeled considering their size as early endosomes (200 nm diameter, EE) and late endosomes (500 nm diameter, LE). In Figures 8 and 9, vacant EE and LE as well as the EE and LE where AuNPs are located were pointed out (blue and red circles, respectively). An average of 15 ± 5 AuNRs/vesicle and 18 ± 6 AuNPrs/vesicle were counted for AuNRs-BSA-A565-r 8 and AuNPr-BSA-A565r 8 , respectively (statistics of 30 vesicles from the TEM images).

Conclusions
In this work, we tested the internalization abilities of two novel nanoconstructs based on anisotropic AuNPs with different surface and charge, AuNRs and AuNPrs coated with BSA in the presence and absence of a CPP (r 8 ), improving AuNP internalization in the presence of r 8 . To the best of our knowledge, this is the first report that describes the coating of AuNRs and AuNPrs (with absorption in the first biological window) with BSA functionalized with r 8 for improved cell internalization. These results point to the ability to improve the biocompatibility and internalization of nanoconstructs into cells by using endogenous proteins and CPPs. Nevertheless, as a further step, it would also be interesting to add targeting agents to the proposed nanoconstructs to increase their selectivity.