Synthesis and characterization of peptide conjugated human serum albumin nanoparticles for targeted cardiac uptake and drug delivery

Congestive heart failure, a prominent cardiovascular disease results primarily from myocardial infarction or ischemia. Milrinone (MRN), a widely used clinical drug for heart failure, improves myocardial contractility and cardiac function through its inotropic and vasodilatory effects. However, lacking target specificity, it exhibits low bioavailability and lower body retention time. Therefore, in this study, angiotensin II (AT1) peptide conjugated human serum albumin nanoparticles (AT1-HSA-MRN-NPs) have been synthesized for targeted delivery of MRN to the myocardium, overexpressing AT1 receptors under heart failure. The NPs were surface functionalized through a covalent conjugation reaction between HSA and AT1. Nanoparticle size was 215.2±4.7 nm and zeta potential -28.8±2.7 mV and cumulative release of MRN was ~72% over 24 hrs. The intracellular uptake of nanoparticles and cell viability was studied in H9c2 cells treated with AT1-MRN-HSA-NPs vs the control non-targeted drug, MRN Lactate under normal, hypoxic and hypertrophic conditions. The uptake of AT1-HSA-MRN-NPs in H9c2 cells was significantly higher as compared to non-targeted nanoparticles, and the viability of H9c2 cells treated with AT1-MRN-HSA-NPs vs MRN Lactate was 73.4±1.4% vs 44.9±1.4%, respectively. Therefore, AT1-HSA-MRN-NPs are safe for in vivo use and exhibit superior targeting and drug delivery characteristics for treatment of heart failure.


Introduction
Cardiovascular disease (CVD) is one of the leading causes of mortality, with incidences constantly on the rise across the developed and developing world. Of these CVDs, myocardial infarction (MI) and congestive heart failure (CHF) are responsible for more than 50% of the global cases of CVDs with high rates of readmission and re-hospitalization [1]. Currently, the most common treatments for CHF include surgical interventions such as heart transplant,

Surface modification of HSA with AT1 peptide
The surface of HSA was modified to facilitate attachment of the AT1 peptide in a two-step reaction. An aqueous solution of 20 mg/mL of HSA dissolved in deionized water (0.3 mM) was prepared and reacted with a 10-fold molar excess of 5(6)-Carboxyfluorescein N-hydroxysuccinimide ester (COOH-FITC-NHS)), for 1 hour. This was further reacted with EDC/Sufo-NHS for 30 minutes followed by reaction with either the AT1 or scrambled peptide (Scr) for 4 hours (Fig 1). The AT1 and Scr peptide was added in a 10-fold molar excess than HSA. The AT1-HSA and Scr-HSA were purified by dialysis using the Slide-a-Lyze dialysis cassette (10,000 Da MWCO). The purified sample was then lyophilized and stored at 4˚C.

Nuclear magnetic resonance
1D proton spectra were recorded at 25˚on a 500 MHz Varian INOVA NMR Spectrometer, with an HCN triple resonance RT probe with Z-axis pulsed field gradients.

Mass spectrometry
The

Nanoparticle preparation
The AT1-HSA-MRN-NPs were prepared by following the ethanol desolvation technique [30,31]. Briefly, an aqueous solution of 20 mg/mL of AT1-HSA was prepared in deionized water. The pH of the solution was adjusted to pH 8.0 using 0.

Nanoparticle characterization, yield and encapsulation efficiency
The average particle size of the nanoparticles was measured by Dynamic Light Scattering (DLS) using a Particle Size Analyzer (Brookhavens Instruments Corporation, NY, USA). The samples were diluted 1:20 with deionized water and measured at a scattering angle of 90˚and temperature of 25˚C. The Polydispersity Index (PDI) estimated the size distribution of the nanoparticles. The zeta potential was measured by a Zeta Potential Analyzer (Brookhavens Instruments Corporation, NY, USA) using electrophoretic laser Doppler anemometry. The size and shape of the nanoparticles were examined by Transmission Electron Microscopy (TEM) (FEI Tecnai G 2 Spirit Twin 120 kV Cryo-TEM, Gatan Ultrascan 4000 4k x 4k CCD Camera System Model 895). The yield of the nanoparticles was measured by the UV-spectrophotometric method. A standard curve of HSA solution dissolved in Bradford reagent was used as a reference and absorbance was measured at 595 nm. To determine encapsulation efficiency, nanoparticles were spin concentrated using centrifugal filters with molecular weight cut off (MWCO) of 10,000 Da for eluting the non-encapsulated MRN into the collection tube. A standard curve of MRN in a mixture containing DDQ/Ethanol was used as a reference and absorbance was measured at 356 nm [32].

In Vitro milrinone release from nanoparticles
The in vitro drug release was studied by UV-visible spectrophotometry 9 . In brief, 40 mg of AT1-HSA-MRN-NPs were suspended in 10 mL of PBS at 37˚C and 120 rpm in a shaking incubator. At predetermined time intervals of 0, 0.25, 0.5, 0.75, 1, 2, 4, 8, 18 and 24 hours, 0.5 mL of the nanoparticle suspension was withdrawn and re-substituted with 0.5 mL of fresh PBS. The withdrawn suspension was centrifuged using Amicon centrifugal filters (10K MWCO) and supernatant was used to determine the amount of MRN released. The MRN was detected at 356 nm using a colorimetric assay and a cumulative MRN release over time was calculated 21 .

Cell culture and viability
Rat cardiomyoblasts (H9c2) cells were received as a kind gift from Dr. Renzo Cecere, M.D. (Montreal General Hospital, QC, Canada). The H9c2 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). H9c2 cells were seeded at an initial density of 5000 cells/well in clear bottom 96-well black plates for 24 hours in a humidified incubator at 37˚C and 5% CO 2 , Cells were treated with AT1-HSA-MRN-NPs, AT1-HSA-NPs, MRN-HSA-NPs and MRN Lactate, diluted in serum-free cell culture medium. The MRN concentration in the nanoparticles and MRN Lactate were 1 mM, as optimized from previous studies [12]. After 4, 24 and 48 hours of incubation, the cells were washed thrice with PBS. Cells were treated with 100 μL of fresh cell culture medium and 20 μL of MTT reagent and incubated at 37˚C and 5% CO 2 for 4 hours. The media was removed, and cells were lysed by addition of 100 μL of DMSO for 15 minutes at room temperature. The absorbance was measured at 570 nm using the Victor3V 1420 Multilabel Counter spectrophotometer.

Overexpression of the AT1 Receptor
The H9c2 cells were seeded at an initial density of 5,000 cell/well in 96-well plates, separated into three groups: Normal, Hypoxic and Hypertrophic. Hypoxia was induced by treatment with 100 μM of CoCl 2 .6H 2 O for 24 hours, which simulated the conditions of MI [33]. Hypertrophy was induced by treatment with 20 μM H 2 O 2 for 48 hours to simulate conditions of CHF [34]. The cells in each group were treated with the anti-AT1 antibody (Abcam, Canada) for 1 hour followed by a goat polyclonal secondary antibody conjugatedto Alexa 488 (Abcam, Canada) for an additional 1 hour, following the manufacturer's recommended dilutions. The cells were fixed with 4% paraformaldehyde in PBS for 10 mins and thrice washed with PBS and stored at 4˚C. The fluorescence was measured at 495 nm excitation/ 519 nm emission wavelengths.

Intracellular nanoparticle uptake
The H9c2 cells were seeded at an initial density of 5,000 cell/well in 96-well plates, separated into three groups: Normal, Hypoxic and Hypertrophic. Cells were subjected to hypoxia by treatment with 100 μM of CoCl 2 .6H 2 O for 24 hours, to simulated MI [33]. Cells were subjected to hypertrophy by treatment with 20 μM H 2 O 2 for 48 hours to simulate HF [34]. The H9c2 cells in each group were treated with 0.5 mg/mL of fluorescently tagged AT1-HSA-MRN-NPs, Scr-HSA-MRN-NPs and MRN-HSA-NPs for 4 hours. The Scr peptide was the same amino acid chain as AT1 peptide but in a scrambled order [20]. The Scr peptide-tagged NPs were used to confirm the targeting efficiency of the AT1 peptide-tagged NPs. The cells were washed thrice with PBS and fresh media was added. The fluorescence intensity was measured at 489nm/535nm using a Victor3V 1420 Multilabel Counter spectrophotometer (Perkin Elmer, Woodbridge, ON, Canada).

Statistical analysis
All statistical analyses were carried out using the GraphPad Prism (v 8.4.3; GraphPad Software, La Jolla, CA) software. One-Way ANOVA with Tukey's multiple comparisons was used to compare the means. All data are expressed as mean ± standard deviation (SD) with n = 3.

Nuclear magnetic resonance
The design and synthesis of the AT1-HSA was initially characterized by 1 H-NMR (Fig 2). The NMR spectrum of AT1 displays peaks at around 6.8 ppm which are due to the presence of tyrosine residues, while most of the downfield resonance is due to histidine. This signal is also visible on the AT1-HSA spectrum. The spectrum contains two sets of signals. The more intense signals arise from the trans peptide bonds whereas the less intense signals are due to a cis-bond between proline and histidine 21 . Peaks at around 4.0 ppm due to glycine residues visible on AT1 spectrum is also visible on the AT1-HSA spectrum. However, the NMR spectra of the HSA-AT1 and HSA in comparison to AT1 peptide displayed broader peaks, which results from the higher molecular weight (66500 Da) of the HSA in comparison with the AT1 peptide (1274 Da). Thus, the data gathered from 1 H-NMR indicate the conjugation of the AT1 peptide with the HSA, which was also confirmed with Mass Spectrometry.

Mass spectrometry
To validate the conjugation of AT1 peptide with HSA, MALDI-TOF-MS was used to compare the average molecular weight change between HSA and AT1-HSA. The mass-to-charge ratio (m/z) of the green peak (AT1-HSA) was approximately 7000 higher than the red peak (HSA). The molecular weights of the AT1 peptide, 5(6)-Carboxyfluorescein-NHS, EDC and Sulfo-NHS is 1274, 376.32, 190 and 217 g/mol, respectively. Upon calculating the cumulative molecular weight of AT1-HSA, and comparing with HSA protein, it can be inferred that at least 3 molecules of AT1 were successfully conjugated with the surface of each HSA molecule (Fig 3).

Nanoparticle characterization
The nanoparticles size was determined using DLS and laser Doppler anemometry for zeta potential analysis. The particle size of AT1-HSA-MRN-NPs was 215.2±4.7 nm with a zeta potential of -28.8±2.7 mV, and size of MRN-HSA-NPs (without AT1) was 189.6±3.8 nm with zeta potential of -27.5±4.6 mV. The morphology of the nanoparticles as observed using TEM under 13500X (Fig 4(A)) and 55,000X magnification exhibited a near spherical shape with  https://doi.org/10.1371/journal.pone.0254305.g004 moderately uniform particle size and even distribution (Fig 4(B)). Under 250,000X magnification, the AT1-HSA-MRN-NPs had a dark core surrounded by a bright membrane, which confirmed the distinct layer (Fig 4(C)). The yield of the AT1-MRN-HSA-NPs was 75.6±2.5%, and encapsulation efficiency was 40.5±1.5%.

In Vitro drug release study
The drug release from AT1-HSA-MRN-NPs was studied by suspending 40 mg of nanoparticles in 10 mL PBS at 37˚C and 120 rpm, with a starting MRN concentration of 0.8 mg/mL prepared from MRN/AT1-HSA (wt./wt.) ratio of 1:10. MRN showed a sustained release with approximately 50% of the MRN released between 4-6 hrs after which the release became slower with up to 75% of the MRN being released by 24 hrs (Fig 5). The drug released at various time points has been presented in Table 1.

Cell viability analysis
The MTT assay was performed for cell viability where H9c2 cells were treated with AT1-H-SA-MRN-NPs, AT1-HSA-NPs, MRN-HSA-NPs and MRN Lactate containing 1mM MRN concentrations for 4, 24 and 48 hours. The 1mM MRN concentration was optimized in our previous study and is being used here for consistency of results [12,35]. The safety analysis of MRN-HSA-NPs was conducted using HUVEC and H9c2 cells in our previous study and therefore was not repeated here [12,35]. Results showed that the H9c2 cells treated with  Fig 6(A)). Compared to the AT1-HSA-MRN-NPs, the cell viability of MRN-Lactate was significantly lower (p = 0.0016). At 24 hours, the cell viability was 55.5±3.7% (p = 0.0055), 70.8±11.2% (p = 0.0509) and 41.2±3.8% (p = 0.0009), respectively compared to the control group (Fig 6(B)), and at 48 hours, 52.4±2.1% (p = 0.0024), 65.9±11.2% (p = 0.0212) and 35.3±7.7% (p = 0.0001), respectively (Fig 6(C)). These results are also summarized in Table 2. Therefore, the AT1-HSA-MRN-NPs exhibit better safety characteristics and lesser cytotoxicity as compared to MRN Lactate. The in vitro cytotoxicity due to hypoxia and hypertrophy treatments was also investigated and compared with normal H9c2 cells. No significant difference in cell viability was observed between the 3 conditions (Fig 7).

Angiotensin II Type 1 receptor overexpression
The AT1R receptor overexpression was studied in hypoxic, hypertrophic and normal H9c2 cells. Results showed that hypoxic and hypertrophic cells exhibited a significantly higher

PLOS ONE
Peptide conjugated human serum albumin nanoparticles for targeted cardiac uptake and drug delivery expression of the AT1 receptors as compared to normal cells (p<0.0001). There was no significant differences between the hypoxic and hypertrophic conditions (p = 0.9883). The fluorescence intensity displayed by the hypoxic and hypertrophic cells was almost twice higher than that observed in the normal cells (Fig 8).

Intracellular nanoparticle uptake analysis
Since AT1 receptors are overexpressed on cardiomyocytes during HF, conjugating the nanoparticles with the AT1 peptide was anticipated to demonstrate higher uptake of the AT1-H-SA-MRN-NPs through receptor-mediated endocytosis. The nanoparticle concentration was   (Fig 9(A)). Similarly, in hypoxic cells, the AT1-HSA-MRN-NPs uptake was significantly higher (p<0.0001) than that of MRN-HSA-NPs and Scr-HSA-MRN-NPs (p = 0.0014) (Fig 9(B)). Also, for

Discussion
The urgent need to effectively treat MI and HF has led to research and innovation of various new strategies and treatment modalities. These include delivery of drugs, growth factors, cytokines and other molecules for myocardial regeneration or treatment [36,37]. However, due to inherent limitations with most treatment strategies such as lack of target specificity, low bioavailability, cardiac rejection while heart pumping, or non-specific distribution, the therapeutic effect is lessened [38]. The emerging studies on nanoparticles and targeted drug delivery systems have displayed promising results, however, their efficacy remains dependent on the drug binding capacity, solubility, nanoparticle degradability and plasma retention time [39]. In this study, keeping in view the intended features of ideal drug delivery systems, a targeted nanoparticle formulation was synthesized. The HSA surface was modified to attach a targeting ligand, AT1 peptide, to achieve superior delivery characteristics. The AT1 peptide shows specificity for the AT1 receptor present on the myocardium, which is found to be overexpressed under CHF conditions [6][7][8].
Using the AT1 peptide as the targeting moiety will facilitate receptor-mediated nanoparticle uptake. Though the angiotensin is a major axis in HF, used for drug-targeting by ACE inhibitors or angiotensin receptor blockers, we are yet to study the study the effect of AT1-HSA-MRN-NPs on receptor activity. However, the targeted delivery of MRN via AT1 receptor-mediated endocytosis of the nanoparticles is aimed at treating HF in an effective manner, thus suppressing exactly these potential effects [40]. The AT1 peptide was conjugated to the HSA surface through a two-step covalent chemical reaction. The 5(6)-Carboxyfluorescein-NHS targets primary amines such as in the side chain of lysine residues, to form stable amide bonds. This allows the carboxylic group to undergo a carbodiimide reaction with EDC, at pH 5.5, forming an unstable amine-reactive O-acylisourea intermediate. This unstable intermediate is further reacted with the Sulfo-NHS and the amine groups on AT1 peptide to release urea as a byproduct and form stable AT1-HSA. Literature suggests that the peptide (NH 2 -Gly-Gly-Gly-Gly-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-NH 2 ) binds with the AT1 receptor over a sizeable binding transmembrane domain (TMD) through interactions between Asp1 (of peptide) with His183 in the second loop of the TMD, Arg2 of peptide with Asp281 in TMD-7, aromatic side chains of Phe8 of peptide with His256, Val254 and Phe259 of the domain, and Tyr4 of the peptide with Asn111 of TMD-3 [41,42].
The pre-modification of the HSA by conjugation with the AT1 peptide was preferred over post-modification of the HSA-NPs as the latter may cause drug loss and leakage during the synthesis and purification. NMR and MALDI-TOF confirmed the binding of AT1 peptide to the HSA surface. In NMR results, HSA shows broad peaks owing to the high molecular weight of 66.5 kDa, whereas the AT1 peptide, with a molecular weight of~1274 g/mol exhibits a clear spectrum showing chemical shifts due to the presence of the amino acid residues. Due to the large differences in molecular weights, the AT1-HSA spectrum resembles the HSA with visible differences at 6.8 and 4.0 ppm indicating AT1 binding. To confirm this, MALDI-TOF-MS was performed. The spectrum on the x-axis represents the m/z (mass/charge) ratio which was 73869.546 for AT1-HSA and 67918.163 for HSA, which was 7000 higher. Considering the mass of the AT1 peptide, the HSA and the cross-linkers, at least 3 AT1 peptide molecules were bound to the HSA surface.
The AT1-HSA-MRN-NPs were formed by the ethanol desolvation process [12,30]. The nanoparticles exhibited a spherical structure as observed under TEM. The AT1-H-SA-MRN-NP size was 215.2±4.7 nm with a zeta potential of -28.8±2.7 mV, in comparison with AT1-HSA-NPs with a size of 189.6±3.8 nm and zeta potential of -27.5±4.6 mV. Typically, particles less than 100 nm size undergo clearance by entering lymphatic capillaries, whereas particles in the size range of 250 nm-1μm are identified by macrophages and removed by the reticuloendothelial system [43,44]. The chances of opsonization are also lowered with reduction in surface curvature. The nanoparticles prepared in this study were in the size range of 100-250 nm with a negative zeta potential, which suggests greater physical stability as nanoparticle aggregation is prevented due to presence of negative charges [45]. The particle size being less than 250 nm suggests a prolonged blood circulation time as the particles will not be rapidly removed through opsonization [46,47]. MRN release over 24 hrs from the nanoparticles further confirmed drug binding to the AT1-HSA-MRN-NPs. The binding characterization of MRN with HSA has been studied extensively in our previous study and was therefore not repeated here [12,35]. Though the release of milrinone has been extensively studied previously with attempts to extend the release over time, only slight improvements have been observed in this area [48,49]. Hence, drug delivery vehicles are needed for effective delivery of MRN to improve the circulation time and rapid clearance from the body along with a targeted approach to further enhance the cardiac uptake and drug activity [40,50].
The in vitro cytotoxicity was evaluated on H9c2 cells treated with MRN-Lactate, AT1-H-SA-MRN-NPs and AT1-HSA-NPs. MRN is clinically administered as a lactate formulation intravenously to adult as well as pediatric patients for HF and associated cardiac conditions [51]. However, the use of MRN Lactate is linked with side effects such as palpitation, cardiac arrythmia and renal dysfunction [52]. This may be attributed to the non-targeted delivery of MRN Lactate and hence the need for a continuous infusion to meet the dosage requirements. Using the targeted nanoparticle formulation, synthesized in this study, as drug carriers this limitation would be overcome, given their higher biocompatibility, higher retention time, drug binding capacity and characteristics of controlled drug release [53]. The cell viability of H9c2 cells was investigated at 4, 24 and 48 hours, with MRN Lactate exhibiting higher cytotoxicity as compared to AT1-HSA-MRN-NPs and AT1-HSA-NPs. The toxicity of the nanoparticles, both HSA-FITC-NPs as well as AT1-HSA-MRN-NPs without fluorescent tagging, has been studied in our previous studies, both in vitro as well as in vivo [12,40]. It was found that the nanoparticles were non-toxic and safe to use in animals and with further testing, will show immense potential for patients. Evaluating nanoparticle toxicity is essential to ensure their suitability for use in future pre-clinical and clinical studies.
The intracellular uptake of the nanoparticles was investigated in normal (non-hypoxic, non-hypertrophic), hypoxic and hypertrophic H9c2 cells and H9c2 cells have been found to be more suitable for cardiac ischemia studies [54]. Inducing hypoxia and hypertrophy in cells closely mimics MI and HF conditions [34,55,56]. The cell viability analysis comparing the normal cell viability with that of hypoxic and hypertrophic H9c2 cells suggested that the hypoxia and hypertrophy inducing treatments were safe and did not cause cytotoxicity. Literature has widely suggested that under HF, the AT1 receptors present on the cardiomyocytes are overexpressed and these receptors could be blocked to reverse cardiac remodeling [7,8]. Therefore, targeting the overexpressed AT1 receptors with the targeted AT1-HSA-MRN-NPs to hypoxic and hypertrophic cardiac cells allowed higher uptake of the AT1-HSA-MRN-NPs as compared to the non-targeted MRN-HSA-NPs and Scr-HSA-MRN-NPs. The Scr peptide-tagged nanoparticles were used as a negative control to ensure that the higher uptake of AT1-HSA-MRN-NPs was a direct result of AT1 peptide mediated targeting and not passive uptake [5]. Also, the uptake of the AT1-HSA-MRN-NPs was significantly higher in hypoxic and hypertrophic cells vs the normal cells. These studies confirm the targeting ability of the AT1 peptide under MI and HF conditions and demonstrate that AT1-HSA-MRN-NPs can safely be used in vivo as targeted drug delivery systems for congestive heart failure and related conditions. In our future studies, we intend to perform in vivo testing using fluorescence imaging to assess the uptake and activity on the drug payload. These studies will include pharmacokinetics, biodistribution and measurement of cardiac function along with toxicity analysis.

Conclusion
To summarize, we have synthesized and developed stable AT1 peptide conjugated albumin nanoparticles to deliver MRN in a targeted manner for heart failure treatment. This novel drug delivery system demonstrates physical stability, release, biocompatibility, specific targeting ability and higher cellular uptake. Also, as compared to the non-targeted MRN Lactate, AT1-HSA-MRN-NPs show greater biocompatibility in cardiomyoblasts. In future, the performance of AT1-HSA-MRN-NPs will be evaluated in a rat model of CHF along with MRN pharmacokinetics. This targeted therapy is anticipated to be more effective in improve the cardiac function in CHF as compared to the currently available treatments.