Cyclic Peptides: Design, Characterization, Self-Assembly, and Applications as Nano Drug Delivery Systems

Over the past two decades, drug delivery systems have become a subject of major interest in the pharmaceutical industry for the treatment of different diseases, such as cancer, viral infections, and genetic disorders. A wide range of compounds show high binding affinity and great potential in in vitro non-cellular assays through direct interacting with molecular targets. However, many compounds show low potency in cell-based assays due to their limited delivery across the cell membrane. The physicochemical properties of several compounds, such as size, poor water solubility, hydrophobic nature, and negative charge, limit their cellular uptake significantly. Thus, there is an urgent need in design and synthesis of novel molecular transporters for efficient delivery of effective compounds to cellular targets. The recent growth of nanotechnology products will expand the current resources of therapeutics for the pharmaceutical industry in the next few years. The use of nanotechnology in drug delivery is widely expected to change the future of the pharmaceutical and biotechnology industries. The application of nanotechnology in drug delivery has led to the discovery of nano Drug Delivery Systems (nano-DDS). Nanocarriers exhibit unique properties and take advantage of specific physiochemical behavior of nanoparticles. Properties such as, magnetism, conductivity, melting and boiling points, and chemical and biological reactivity become different at nano scale due to the quantum mechanical behavior of extremely small structures at molecular dimensions. Furthermore, nanoparticles behave differently since they take advantage of extraordinary high surface area to mass ratio, leading to increased surface interactions and distinct biological performance. Nanoparticles have the potential to be manipulated through changing their size, electronic, and hydrophobic nature. Employing nanosized carriers offer several advantages, including enhanced intracellular delivery of poorly water-soluble drugs, targeted delivery, transporting relatively large biologically important molecules across the cell membrane, delivery of a combination of drugs to overcome drug resistance, and transporting drugs through challenging epithelial and endothelial barriers. Thus, design and development of nano-sized pharmaceutical carriers has become attractive for chemists, biologists, and physicists. Functionalized Nano-DDS are designed to deliver and release cargo drugs intracellularly more efficiently than the currently available systems, leading to the enhanced drug tissue bioavailability and eventually therapeutics efficacy. Among all nano-DDS, Cell-Penetrating Peptide (CPP)-mediated intracellular DDS has been widely used for the enhanced delivery of water insoluble drugs, negatively charged molecules (e.g., DNA, siRNA, phosphopeptides), and proteins. However, the application of (CPP)-mediated intracellular DDS in in vivo models has been challenging due to their inherent toxicity to normal cells and organs. Several studies have been performed to facilitate the intracellular delivery of a wide range of low-molecular weight and macromolecular drugs using carriers through the cell cytoplasm bypassing the endocytic pathway to avoid lysosomal degradation. In this process, although the drug is delivered into cytoplasm protected, it still needs to be transported to a certain organelles, such as mitochondria or nuclei, where therapeutic function occurs. Several CPPs promote the delivery of drugs through receptormediated endocytosis. However, this mechanism requires high affinity between the drug-carrier complex and the target in the cell membrane for endocytosis. This process will be followed by energy-dependant formation of endosomes. However, this method suffers from entrapment and release challenges. After delivery of the molecular cargo into cells through endocytic pathway, and its entrapment in endosome, the cargo molecules or drug may end up in lysosome and degraded by the lysosomal enzymes. Thus, a limited amount of the drug can reach the cytoplasm because of inadequate endosomal escape and lysosomal degradation. As a result, although numerous CPPs exhibit promising results in in vitro assays, they fail in vivo because of the poor bioavailability. Furthermore, the nuclear delivery of cargo drugs with known CPPs has been unsuccessful. The nucleus of a cell is an important target for drug delivery systems, due to presence of the genetic information and transcription machinery. An efficient cellular uptake of the drug is highly desired in nucleus where it can interact with nucleic acids. The nuclear targeting delivery is challenging. The designed drug-CPP complex must meet several requirements for nuclear delivery including efficient cell internalization by receptor-mediated endocytosis (RME), escaping endosomal/lysosomal pathways, acquiring a nuclear localization signal to communicate with nuclear pore complexes, and eventually, being sufficiently small to enter the nuclear membrane. Not many CPPs have been found efficient for the nuclear delivery applications. CPPs can be classified into two major classes, linear and cyclic CPPs. Most reported CPPs are linear peptides that are flexible in solution and contain up to 10 amino acids. However, cyclization of peptides has been employed as a strategy to generate constrained structures. The rigidity imposed by cyclization reduces the flexibility and causes the system to adopt a restricted numbers of molecular conformations. Peptide cyclization has become a unique approach to generate conformations not available to linear peptides. Cyclic and linear peptides containing an equal number of similar amino acids create different geometries leading to different affinities for similar targets. Furthermore, cyclic CPPs containing specific amino acids have shown to have a different cellular uptake mechanism compared to linear CPPs. While most of the linear CPPs undergo endocytosis pathways in cellular uptake, some of the cyclic CPPs have endocytosis-independent uptake and target nucleus. Thus, cyclic peptides can be designed to be used as nuclear delivery vehicles of anticancer compounds targeting DNA. Functionalizing cyclic peptides with tumor targeting moieties can be used as a strategy for selective cancer cell targeting and improving nuclear targeting of anticancer drugs. Cyclic CPPs can be also covalently conjugated to active drug cargos to generate prodrugs. Prodrugs are chemically modified analogs of an active metabolite that can improve pharmacokinetics and pharmacodynamics (PK/PD) properties of the drug. Prodrug strategy could offer several advantageous, such as enhancing water solubility, drug delivery, and chemical stability, and reducing toxicity. However, chemical transformation to the active drug is required in the presence of different intracellular enzymes to convert prodrugs to their corresponding pharmacologically potent moieties in in vivo systems. Cyclic peptide-drug conjugates can be used as alternative prodrug approach for improving delivery of compounds with limited

differently since they take advantage of extraordinary high surface area to mass ratio, leading to increased surface interactions and distinct biological performance.
Nanoparticles have the potential to be manipulated through changing their size, electronic, and hydrophobic nature. Employing nanosized carriers offer several advantages, including enhanced intracellular delivery of poorly water-soluble drugs, targeted delivery, transporting relatively large biologically important molecules across the cell membrane, delivery of a combination of drugs to overcome drug resistance, and transporting drugs through challenging epithelial and endothelial barriers.
Thus, design and development of nano-sized pharmaceutical carriers has become attractive for chemists, biologists, and physicists. Functionalized Nano-DDS are designed to deliver and release cargo drugs intracellularly more efficiently than the currently available systems, leading to the enhanced drug tissue bioavailability and eventually therapeutics efficacy.
Among all nano-DDS, Cell-Penetrating Peptide (CPP)-mediated intracellular DDS has been widely used for the enhanced delivery of water insoluble drugs, negatively charged molecules (e.g., DNA, siRNA, phosphopeptides), and proteins.
However, the application of (CPP)-mediated intracellular DDS in in vivo models has been challenging due to their inherent toxicity to normal cells and organs. Several studies have been performed to facilitate the intracellular delivery of a wide range of low-molecular weight and macromolecular drugs using carriers through the cell cytoplasm bypassing the endocytic pathway to avoid lysosomal degradation. In this process, although the drug is delivered into cytoplasm protected, it still needs to be transported to a certain organelles, such as mitochondria or nuclei, where therapeutic function occurs. Several CPPs promote the delivery of drugs through receptormediated endocytosis. However, this mechanism requires high affinity between the drug-carrier complex and the target in the cell membrane for endocytosis. This process will be followed by energy-dependant formation of endosomes. However, this method suffers from entrapment and release challenges. After delivery of the molecular cargo into cells through endocytic pathway, and its entrapment in endosome, the cargo molecules or drug may end up in lysosome and degraded by the lysosomal enzymes.
Thus, a limited amount of the drug can reach the cytoplasm because of inadequate endosomal escape and lysosomal degradation. As a result, although numerous CPPs exhibit promising results in in vitro assays, they fail in vivo because of the poor bioavailability.
Furthermore, the nuclear delivery of cargo drugs with known CPPs has been unsuccessful. The nucleus of a cell is an important target for drug delivery systems, due to presence of the genetic information and transcription machinery. An efficient cellular uptake of the drug is highly desired in nucleus where it can interact with nucleic acids. The nuclear targeting delivery is challenging. The designed drug-CPP complex must meet several requirements for nuclear delivery including efficient cell internalization by receptor-mediated endocytosis (RME), escaping endosomal/lysosomal pathways, acquiring a nuclear localization signal to communicate with nuclear pore complexes, and eventually, being sufficiently small to enter the nuclear membrane. Not many CPPs have been found efficient for the nuclear delivery applications. Cyclic CPPs can be also covalently conjugated to active drug cargos to generate prodrugs. Prodrugs are chemically modified analogs of an active metabolite that can improve pharmacokinetics and pharmacodynamics (PK/PD) properties of the drug. Prodrug strategy could offer several advantageous, such as enhancing water solubility, drug delivery, and chemical stability, and reducing toxicity. However, chemical transformation to the active drug is required in the presence of different intracellular enzymes to convert prodrugs to their corresponding pharmacologically potent moieties in in vivo systems. Cyclic peptide-drug conjugates can be used as alternative prodrug approach for improving delivery of compounds with limited cellular uptake.
This dissertation focuses on a class of cyclic peptides as intracellular molecular transporters that can be used as prodrugs or peptide-capped gold nanoparticles. We hypothesized that the combination of alternate hydrophobic tryptophan and positively charged arginine or lysine residues in the sequence of the cell-penetrating cyclic peptide was critical for improving the cell-penetrating properties of the system.
Furthermore, the presence of arginine and tryptophan facilitated the formation of gold nanoparticles. The molecular transporting efficiency of the peptide alone and peptidecapped gold nanoparticles was evaluated for a broad range of molecular cargos including anti-HIV drugs, anticancer agents, and negatively charged phosphopeptides.
The cyclic peptides containing alternate tryptophan and arginine residues and their corresponding peptide-capped gold nanoparticles enhanced the delivery of water insoluble drugs and negatively charged biologically important molecules. A broad range of parameters including concentration, toxicity, time, and different sequences of amino acids were explored to optimize the cellular uptake. Several characterization methods were used to determine the interaction between drugs and carrier through the formation of the complex. Different methods were also used to investigate the mechanism of the cellular uptake.
This work presents examples where prodrugs containing linear peptides are compared with their corresponding cyclic peptides in terms of biological properties and in delivery of different drugs. For example, cyclic peptides containing certain amino acids generated gold nanoparticles more efficiently compared to their corresponding linear peptides. Moreover, cyclic peptide capped gold nanoparticles exhibited higher molecular transporting potency when compared with the linear counterparts. This dissertation will be discussed in four manuscripts: The objective of Manuscript І (published in Molecular Pharmaceutics 2013, 10(5), [2008][2009][2010][2011][2012][2013][2014][2015][2016][2017][2018][2019][2020] was to evaluate a cyclic octapeptide containing arginine and tryptophan for their ability to transport negatively charged phosphopeptides.
Phosphopeptides are important compounds for studying protein-protein interactions.
Phosphopeptides suffer from their limited cellular uptake due to the presence of negatively charged phosphate group in their structure. The hypothesis of this manuscript is that cyclic peptide [WR] 4  However, this strategy was required to be evaluated for non-cell penetrating peptides.
Thus, we used a sequence of amino acids for designing a non-cell-penetrating peptide that generated peptide-capped gold nanoparticles with molecular transporting property.
In Manuscript ІV, (published in Molecular Pharmaceutics, DOI: 10.1021/mp400199e), a green method for the synthesis of non-cell penetrating peptide-capped gold nanoparticles was reported. We hypothesize that a non-cell penetrating peptide containing lysine and tryptophan namely linear (KW) 5 and cyclic [KW] 5 can be converted to a cell-penetrating molecular transporter upon generation of cyclic peptide-capped gold nanoparticles (linear (KW) 5    In summary, the studies described in this dissertation provided insights about the application of a new generation of cyclic peptides for the delivery of a broad range of drugs and biomolecules. The cyclic peptide containing alternate tryptophan and arginine residues was found to be efficient molecular transporters and nuclear delivery agents ( Figure 1). In addition, we have demonstrated that a non-cell penetrating cyclic peptide can be converted to an efficient intracellular drug transporter through generation and capping of the gold nanoparticles. A detailed investigation on the cyclic nature of the peptide compared to the linear one in terms of their chemical behavior and biological activity was provided. Overall, a concrete strategy has been established by developing non-toxic cyclic peptide molecular transporters that can be used in prodrug strategy or gold nanoparticle formation. The scientific knowledge was advanced in the area of application of cell-penetration peptides in prodrug strategy and designing nanocarriers.

MANUSCRIPT Ш
There are no Schemes in Manuscript Ш

MANUSCRIPT ІV
There are no Schemes in Manuscript ІV

Introduction
Peptide amphiphiles (PAs) are composed of a combination of amino acids carrying hydrophobic/hydrophilic and positively/ negatively charged residues. 1 PAs have been found to be promising tools in drug and gene delivery due to their biocompatibility and bioactivity. 2−4 These compounds are not toxic to cells at their experimental concentration and are able to cross the cell membrane because of their unique structural properties.
Hydrophobic unit of PAs can contain long chain fatty acids or amino acids with hydrophobic side chain residues. This unit generates a pocket that could be responsible for the entrapment of drugs and facilitates the internalization into the cell membrane. 5 At the same time, the presence of positively charged arginine facilitates the interaction between the peptide and the negatively charged phospholipids on the cell membrane. The application of positively charged linear cell-penetrating peptides (CPPs) as drug carriers for biologically active cargos has been reported previously. 6−9 Polyarginines, TAT (trans-acting activator of transcription), and Penetratin have been found to enhance the cellular uptake of a diverse range of drugs. 10 23 Peptidomimetic prodrugs have been reported for intracellular delivery of aryl phosphoramidates 24 and difluoromethyl phosphonates. 25 Arrendale and co-workers developed a (difluoromethylene)phosphoserine prodrug to deliver negatively charged phosphoserine peptidomimetic intracellularly. FOXO3a is a transcription factor that is phosphorylated by Akt1 and binds to 14-3-3-adaptor protein. The released modified phosphopeptide was able to release FOXO3a from 14-3-3 protein, leading to cell death in leukemia cells. 26,27 Other phosphoserine and phosphothreonine masked phosphopeptides have been evaluated in studying kinase regulation. 28,29 Considering the critical roles of the phosphopeptides in cellular signaling pathways, we have previously reported amphiphilic linear peptide analogs to improve the intracellular uptake of negatively charged phosphopeptides through noncovalent   utilizing GAUSSIAN'03 software. Geometry of monomers was completely optimized.
All bond lengths, bond angles, and dihedral angles were allowed to relax without any constraint. The equation for three sequential binding events was found to be the best fit for the data as shown by the Origin software. The chi squared χ2 value was found to be 1.6 ×

Conclusions
In summary, a new carrier system for the delivery of cell impermeable and PEpYLGLD was further confirmed by ITC studies. To the best of our knowledge, this is the first report of using cyclic peptides for noncovalent cellular delivery of cell impermeable phosphopeptides. These data provide insights about using cellpenetrating cyclic peptides as PAs for delivery of negatively charged cell impermeable phophopeptides.

Supporting Information
Analytical HPLC data, high resolution mass spectra, and additional supporting data.
This material is available free of charge via the Internet at http://pubs.acs.org.

Notes
The authors declare no competing financial interest.

ACKNOWLEDGMENTS
We acknowledge the financial support from National Science Foundation,   dose of Dox is required in cancer chemotherapy to achieve a sufficient therapeutic effect, which leads to dose-dependent side effects, such as such as cumulative cardiotoxicity, nephrotoxicity, and extravasation. 11,12 Moreover, intracellular Dox accumulation is dependent on a number of factors including cellular uptake, retention, relocalization, and efflux from the cell. Among 46 these factors, intracellular uptake of Dox suffers from efflux pumping in some cancer cells, such as ovarian carcinoma cells, leading to decreased intracellular Dox levels that could be related to the overexpression of energy-dependent drug efflux pump proteins such as P-glycoprotein (P-gp). This integral membrane protein removes drugs and thus reduces intracellular anticancer drug concentrations. 13 Efficacy and toxicity of an anticancer drug can be modified through using drug delivery systems and altering the physicochemical properties, such as lipophilicity, cellular uptake, and prolonging activity through chemical conjugation with various chemical moieties. One of the main applications of drug delivery systems is to avoid the P-gp and multidrug resistance proteins (MRPs) that are involved in drug efflux to overcome the resistance problem and P-gp-mediated drug efflux. 14−16 Prodrug strategy as one of the drug delivery systems through chemical conjugation with a parent drug 17,18 has been widely used in Dox delivery. 6 Herein, we report the synthesis of linear and cyclic peptides through the covalent conjugation with a 3-carbon chain linker at the 14-hydroxyl group, evaluation of their in vitro anticancer activities in multiple cancer cell lines, and cellular uptake and retention. The prodrug conjugates were designed to improve cellular uptake, to prolong biological activity, and to reduce intrinsic cellular efflux of Dox.  The area under the curve (AUC) was calculated and used to find out the percentage of released Dox and remaining prodrug at a given time. Figure S1 Figure S4).

Supporting Information
Materials and methods, general chemistry, mass spectra, and additional supporting data and figures for stability and cell cycle arrest. This material is available free of charge via the Internet at: http://pubs.acs.org.

Notes
The authors declare no competing financial interest.           moieties has been also investigated as an efficient strategy for drug delivery. 8 We have previously reported the application of a number of cyclic peptides as nuclear targeting molecular transporters. 9 Herein, we report evaluation of L-cyclic peptides as simultaneous reducing and capping agents for generation of cyclic peptide-capped gold nanoparticles (CP AuNPs). We used cellpenetrating properties of both cyclic peptides and capped AuNPs for in situ generation of a DDS. To the best of our knowledge, this is the first report of using cyclic peptides in generation of cellpenetrating CP-AuNPs and as drug delivery tools.

ACKNOWLEDGMENTS
Multifunctional CP-AuNPs with Au 3+ reducing, capping, and cell-penetrating properties are distinct from the previously reported AuNPs for the following reasons: Furthermore, positively charged linear peptides found to enhance the reduction because of the favorable charge interactions can bring the chloroaurate anions in proximity for reaction. 13 The application of cell-penetrating cyclic peptides as simultaneous reducing and capping agents in AuNP formation remains unexplored. (4) The enhancement of intracellular delivery of biologically active cargos by employing linear cationic cell-penetrating peptides (CPPs) has been previously reported. 14,15 Compared to linear peptides that are susceptible to hydrolysis by endogenous peptidases, cyclic peptide counterparts are chemically and enzymatically more stable. 16 (5) CP-AuNPs in combination with antiviral and anticancer drugs can be used as potential scaffold for generation of noncovalent prodrugs.

EXPERIMENTAL SECTION
Materials and Methods. The synthesis of cyclic peptides was carried out according to the previously reported procedure. 7 All reactions were carried out in Bio-Rad polypropylene columns by shaking and mixing using a Glass-Col small tube rotator in dry conditions at room temperature unless otherwise stated. Chlorotrityl chloride resin, trityl chloride resins, coupling reagents, and Fmoc-amino acid building blocks were purchased from Chempep (Miami, FL).
Other chemicals and reagents were purchased from Sigma-Aldrich Chemical Co.
(Milwaukee, WI). All peptides were synthesized using a solid-phase synthesis method  Furthermore, UV−vis spectroscopy study was carried out using HAuCl 4 (1 mM) and [WR] 4 (100 μM to 2 mM) on a 96-well plate, and the absorbance was read after 4 h incubation using a SpectraMax M2 spectrophotometer to determine the optimized concentration ratio for the reduction. The visible range was chosen because of the characteristic surface plasmon peak of AuNPs appearing around 520−550 nm. All experiments were performed in triplicate.

DISCUSSION
AuNPs are usually synthesized under sometimes harsh conditions in the presence of Au(III) ions and inorganic (e.g., sodium borohydride, hydrazine) or 111 organic (e.g., sodium citrate, ascorbic acid) reducing agents to control the size and shape of particles. It has been previously reported that AuNPs generated by peptides exhibited less toxicity compared to chemical methods. 13  Flow cytometry studies showed the cellular uptake of the corresponding fluorescence-labeled CP-AuNPs to be rapid even after 10 min in CCRF-CEM cells.
Inductively coupled plasma mass spectrometry further confirmed the intracellular presence of gold after incubation of [WR] 4 -AuNPs in SK-OV-3 cells. The mechanism of cellular uptake is under investigation, but it is presumably through cellular uptake of AuNPs and the interactions of positively charged and hydrophobic residues of the cell-penetrating peptide 9 with the corresponding negatively charged and hydrophobic 112 moieties in the phospholipid bilayer. Both the peptide and AuNPs have cellpenetrating properties. We previously reported that the cyclic peptides can act as nuclear targeting molecular transporters. 9 Cyclic peptide-capped AuNPs presented a new complex system including both AuNPs and the cyclic peptide.

Notes
The authors declare no competing financial interest.

ACKNOWLEDGMENTS
We acknowledge the financial support from the American Cancer Society, Grant No. Although NPCs operate as available passages for all exchange between the nucleoplasm and cytoplasm, they can be exploited as pathways for nanoparticle delivery. 5,6 Therefore, the delivery of the therapeutic agents to the nucleus by using nano-DDS offers several advantages compared to other systems.

RSG
The application of prodrug strategy among researchers is now well established.
Prodrugs are chemically modified analogs of the active metabolite that can improve pharmacokinetics and pharmacodynamic (PK/PD) properties of the active drug.
However, intracellular chemical transformation needed to be occurred in the presence of different enzymes to convert prodrugs to their corresponding pharmacologically potent compounds in in vivo systems. Prodrug approach offers several advantageous, such as enhancing water solubility, improved chemical stability, decreased toxicity, and insufficient brain penetration. 7 Gold nanoparticles (AuNPs) have received an increased attention due to their unique potential use as nano-DDS. 8 Most of the known AuNPs as nano-DDS lead to the intracellular localization of nanoparticles mainly in the cytoplasm. 9 However, appropriate functionalization of AuNPs with other compounds can be used for their optimization as nano-DDS for a specific application.
Peptide-mediated drug delivery has been widely used for a broad range of cargo molecules including drugs, siRNA, and genes due to their versatility and the presence of a wide range of amino acids. 10 Thus, the peptides have the potential to be used for decoration of the surface of metal nanoparticles. Recently, AuNPs functionalized by peptides have been used as biocompatible systems in drug delivery. 11 For example, AuNPs capped by linear Tat was reported to be able to get internalized into the cytoplasm of 3T3 and HepG2 cells. 11c After the localization of DDS in the destination, it should release the cargo.
Thus, drug loading and release issues are critical for optimized DDS function. Two major strategies for drug loading include covalent and non-covalent binding between AuNPs and drugs. 12 Non-covalent loading offers two main advantages, including ease of the drug loading and facilitating drug release in intact form over covalent loading. 13 Thus, the entrapment of unmodified drugs into functionalized AuNPs through noncovalent interaction is introduced as one of the prodrug strategies for the delivery of a broad range of drugs. Employing appropriate surface functionalization provides a hydrophobic pocket for the loading of drugs. 131 We have previously reported cyclic peptides containing alternative arginine (R) and tryptophan (W) as efficient molecular transporters that showed improving the cellular uptake of different cargos. 14 Subsequently, it was found that a physical mixture of the cell-penetrating cyclic peptide [WR] 4 and HAuCl 4 led to the formation of peptide-capped gold nanoparticles (P-AuNPs) and enhanced the cellular uptake of drugs significantly. 15 It remains to be determined whether non-cell penetrating peptides can be converted to a nuclear targeting DDS through generation of peptide-AuNPs.
Herein, we report the generation of a novel nuclear-targeting nano-DDS containing AuNPs and a non-cell penetrating peptide containing alternative lysine and tryptophan residues. The presence of these amino acids was found to be appropriate to generate in situ biocompatible AuNPs. To the best of our knowledge, this is the first report of converting a non-cell penetrating peptide to an efficient nuclear targeting nano-DDS through cyclic peptide-capped AuNPs formation.

General
All

Synthesis of Fluorescence Labeled-A-Cyclic[KWKWKWKWKW] (F-[KW] 5 ).
The (1 mM) using 96-well plate, and the absorbance was read using SpectraMax M2 spectrophotometer (Molecular Devices, CA) ( Figure S16). The visible range was chosen because of the characteristic maxima peak of AuNPs appeared around 520-560 nm. All experiments were performed in triplicate. The loading efficiency was calculated using the following equation:

Encapsulation of Camptothecin (CPT
To measure the loading capacity, the amount of the AuNPs in the dialysis membrane was measured after 24 h by using ICP-MS. The ICP-MS results showed the amount of AuNPs were responsible for the encapsulation of Dox. Thus, the loading capacity was calculated based on the following equation.  To optimize the yield of P-AuNPs by increasing the reducing and capping efficiency of the peptide, cyclic c[KW] 5 containing ten alternative tryptophan and lysine ( Figure   1) was synthesized. The corresponding linear peptide l(KW) 5 (Figure 1)  The CD spectra demonstrated a significant decrease in spectral elipticity of both P-AuNPs compared to their corresponding parent peptides, suggesting significant modification of secondary structures upon reducing Au 3+ and/or binding to AuNPs.

Transmission Electron
These data show the binding between peptides and AuNPs leads to the formation of c[KW] 5 -AuNPs and l(KW) 5 -AuNPs with differential CD spectra pattern compared to the parent peptides.

Encapsulation of Camptothecin by Peptide-AuNPs
To evaluate the potential of c[KW] 5 -AuNPs and l(KW) 5  Moreover, the intensity of the maxima band was decreased that could be due to the self-quenching of bound-drug and/or the result of the partitioning and entrapment in the hydrophobic pocket generated by the P-AuNPs. 20 These data exhibited that the P-AuNPs were able to encapsulate CPT due to the generated hydrophobic pocket possibly by tryptophan in the structure of the peptide. versus the corresponding linear system (F-l(KW) 5 -AuNPs) as shown by overlayed picture with DAPI ( Figure 6).
Cells use different mechanisms to internalize macromolecules and particles, such as phagocytosis, micropinocytosis, and receptor-mediated endocytosis (RME) pathways including clathrin-mediated, caveolae-mediated, and caveolae/clathrin independent endocytosis. 21 To get a better understanding of the mechanism of peptide- As it is shown in Figure 7, the intracellular uptake of F-l(KW) 5 -AuNPs and Fc[KW] 5 -AuNPs did not significantly change in SK-OV-3 cells in the presence of different endocytic inhibitors after 1 h incubation, suggesting that the mechanism of cellular uptake is not exclusively clathrin-mediated or caveolae-mediated endocytosis, and macropinocytosis. These peptide-AuNPs provide an advantage to known gold nanoparticles and cell-penetrating peptides that their uptake is dependent mainly on endocytotic entry. 22 The surface decoration of AuNPs by amphipathic c[KW] 5 and l(KW) 5 peptides could improve the interactions of lysine and tryptophan residues with the corresponding negatively charged phospholipids and hydrophobic residues in lipid bilayer. This interaction could be a strong driving force for the initial entry into the cell membrane. Hydrophobic interactions generated by tryptophan residues and the 153 lipids can potentially distort the outer phospholipid monolayer. This process will be followed by peptide internalization and enhanced cellular uptake of the cargo. The nature of the peptide in surface of AuNPs is an important parameter that can alter the mechanism of nanoparticle uptake by cells. Further investigation is required to pinpoint the detailed mechanism of cell entry by these P-AuNPs. suggesting that the uptake of drugs is facilitated by P-AuNPs. The results showed that parent cyclic or linear parent peptides did not improve the cellular uptake of drugs.
However, after the formation of P-AuNPs, the cellular uptake of drugs was increased significantly. c[KW] 5 -AuNPs were found to be more efficient transporter than l(KW) 5