Gold Nanoparticle-Small Drug Molecule Conjugates: Therapeutic Applications and Benefits as Compared to Free Drug

 

Pravin Kumar, Mahendra Singh Ashawat, Vinay Pandit

Department of Pharmaceutics, Laureate Institute of Pharmacy, Kangra, H. P, India-177101

*Corresponding Author E-mail: kumar3785@gmail.com

 

ABSTRACT:

Gold nanoparticles (AuNPs) have been profoundly investigated for drug delivery applications. Various widely used small drugs molecules when conjugated either covalently or non-covalently with the AuNPs, showed improved therapeutic effect with reduced dose and adverse effects as compared to drug alone. This review focuses on therapeutic efficiency of AuNPs-small drug molecule conjugates as anti-cancer, anti-bacterial, anti-retroviral and anti-arthritic agents. The basic approaches of conjugation, brief of procedure followed for activity determination and mechanism of action of conjugates have also been discussed.

 

KEY WORDS: Gold, Nanoparticles, Drug conjugates, Anti-cancer, Anti-bacterial, Drug delivery.

 

 


INTRODUCTION:

Michael Fraday, in the year 1957 first described the gold nanoparticles (AuNPs), as colored solution formation on reduction of gold chloride in presence of sodium citrate.1Since that time, AuNPs have been developed by different techniques and investigated for different purposes in medical science. Gold nanoparticles have been exhaustively investigated and established for its use in several fields of medical science viz. immune diagnostics, DNA diagnostics and biosensors etc.2 Further, AuNPs have been investigated for application as anti-neoplastic, anti-arthritic, antibacterial and anti-diabetic agent. Hirsch et al. reported the use of AuNPs in thermal ablation therapy of tumors.3 Christopher et al. and Leonaviciene et al. have studied the anti-arthritic property of colloidal gold in animal models.4, 5Li et al. have reported the use of AuNPs as very efficaous antibacterial at low dose against multi drug resistant pathogens.6 Venkatachalam etal. have reported the significant anti-diabetic effect of AuNPs in alloxan induced diabetic male albino rats.7

 

Received on 10.10.2017          Accepted on 18.12.2017         

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2018; 8 (1): 52-62.

DOI: 10.5958/2231-5713.2018.00009.0

Daisy et al. have reported the anti-diabetic property of AuNPs on streptozotocin induced adult male albino wistar rats.8

 

The important advantage of AuNPs is that it can be synthesized easily in various shapes viz. spherical, rod shaped, core shell and many forms with varied sizes from 1nm to more than 100 nm.9 AuNPs have been considered as an useful delivery vehicles for the different therapeutic agents. These agents include small drug molecules and large macromolecules such as protein, peptides, DNA and RNA.10 Large functional surface to mass ratio enables the AuNPs to be conjugated with different therapeutic agents. The most important step in conjugating  a drug to the AuNPsis the functionalization of nanoparticles. Bhumkar et al. has reported chitosan fuctionalized AuNPs as carrier for insulin.11 Oshi et al. conjugated the thiolatedsi RNA with AuNPs for cellular delivery in HuH-7 cells.12 Wang et al. reported the delivery of plasmid DNA by attaching β-cyclodextrin on the boundry of oligo (ethylene diamino) fuctionalized AuNPs for the treatment of breast cancer.13

 

Presently, AuNPs-drug conjugate are of at most interest, owing to improved treatment efficiency with reduced dose and adverse effects. There are several approaches of conjugating the drugs to AuNPsviz. covalent binding, encapsulation, electrostatic absorption, and other non-covalent assemblies.10 However, the fundamental purpose of writing this review is enable readers to understand that the efficiency of some important and widely used drugs can be improved, when covalently or non-covalently conjugated with the AuNPs.

 

 

Therapeutic application of drug conjugated gold nanoparticles:

Cancer chemotherapy:

Gold nanoparticles (AuNPs) conjugated with anticancer drugs had been widely investigated and reported for the treatment of cancer. A variety of anticancer drugs such as cisplatin, doxorubicin, tamoxifen, paclitaxel etc have been covalently conjugated with the AuNPs (Table 1).

 


Table 1. Gold nanoparticles covalently or non-covalently conjugated with anti-cancerous drugs

Drug

Linking agent between AuNP and Drug

Type of bond

Reference

Cisplatin

Oligonucleotide

Covalent

17

Diamino (polyethylene glycol)

Covalent

18

Cisplatin and folic acid

Mercapto-polyethylene glycol (PEG)

Cisplatin to AuNPs-Covalent,

Folic acid to cisplatin conjugated AuNPs- Non Covalent

19

Oxiplatin

Thiolated-PEG

Covalent

20

Metformin

Hyaluronic acid

Covalent

21

Chloroquine

6-mercaptoundecnoic acid

Covalent

22

5-aminolevulinic acid

-

Covalent

23

Methothrexate

-

Covalent

24

6-Mercaptopurine

-

Covalent

26

Paclitaxel

Thiolated oligonucleotide

PEG and rhodamine B linked β-cyclodextrin

Covalent,

Inclusion complex of paclitaxel and β-cyclodextrin

29

30

Doxorubicin

Thiotic acid-PEG

Covalent (Hydrazone bond)

31

Thioalkyl tetra (ethylene glycol)lyatedtrimethyl ammonium or thioalkyl tetra (ethylene glycol) lyated carboxylic acid

Covalent

33

5-Fluorouracil

Mixed photocleavable and zwitter ionic thiolated alkyl tetra (ethyleneglycol) ligands

Covalent

35

Tamoxifen

Thiolated PEG

Covalent

36

Alkanethiol interior and a tetra (ethylene glycol) hydrophilic shell, terminated with a zwitterionic head

Entrapment in interior hydrophobic pocket

38

Khalalide F

Sodium citrate capped

Covalent

37

β-lapachone

Alkanethiol interior and a tetra (ethylene glycol) hydrophilic shell, terminated with a zwitterionic head

Entrapment in interior hydrophobic pocket

38

Pc4

PEG

Covalent

39,40

 


AuNPs and platinum based conjugate:

Investigators have formulated and studied, a prodrug, platinum (IV)-AuNPs  conjugate to deliver platinum (II) intracellular for cancer therapy.14-16 Intracellularly platinum (IV) undergoes reduction to platinum (II) by the action of glutathione. Platinum (IV) is having low reactivity with biological agents as compared to platinum (II). Thus, conjugation of Platinum (IV) with AuNPs, leads to higher systemic availability and reduced toxicity of platinum (II) to non-cancerous cells.17 In a study, Lippard et al. formulated stable AuNPs, functionalized with oligonucleotide having terminaldodecyl amine groups and attached platinum (IV) complex having carboxyl group using standard carbodiimide coupling chemistry with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxy succinimide sodium salt (NHS)(Fig 1).The anticancer activity of conjugate on variety of cancerous cells has been reported to be either equal or more than the cisplatin alone. Although, in most cases the activity of complex (IC50) is almost similar as compared to unconjugated cisplatin. But, still it can be considered as an advantage because of more efficient delivery of drug to cancer cells with less number of side effects.17 Min et al. using a similar approach synthesized platinum (IV)-gold nanorodspro drug.  Gold nanorods were first covalently functionalized with a diamino (polyethylene glycol) by using in situdithio carbamate formation on one terminal and using the secondamino group for coupling with a carboxyl-containing platinum(IV) compound on other terminal. In vitro cytotoxicity determination by MTT assay on three different cancerous cell lines indicated a higher toxicity compared to free cisplatin.18



Fig 1. Coupling of Pt(IV) prodrug to oligonucleotide functionalized AuNPs.18

 


Patra et al. has reported the fabrication of mercapto-polyethylene glycol (PEG-SH) functionalized AuNP conjugated with cisplatin and folic acid for treatment of ovarian cancer. Biological functional characterization using in vitro proliferation assay showed a time and dose dependent effect of cisplatin on OV-167 cells either as free drug or conjugated system. But, one advantage with the conjugate was observed that it retains the cytotoxic effect of cisplatin but, also protects the normal cells from the cytotoxic insult. Further, the conjugate having folic acid is a suitable vehicle for drug targeting due to the over expression of folate receptors in ovarian cancer cells.19Brown et al. synthesized and investigated a thiolated poly ethylene glycol functionalized AuNPs, tethered with the active component of oxaliplatinfor improved drug delivery in cancer. The platinum tethered nanoparticles showed either almost similar or significantly better cytotoxicity to lung epithelial (A549) and colon (HCT116, HCT15, RKO and HT29) cancer cell lines as compared to free oxaliplatin. The conjugates showed the ability to penetrate within the nucleus of lung cancerous cell lines.20

 

AuNPs and organic micromolecules conjugates:

Another important widely investigated approach to improve the cancer therapy is the AuNPs-organic micromolecules conjugate systems. Small organic molecules such as metformin, paclitaxel, doxorubicin, 5-fluorouracil, tamoxifen, methotrexate (MTX) etc either having a reactive moiety or has been modified to link them with AuNPs.

 

 

 

Senthil Kumar et al. have synthesized hyaluronic acid (HA) functionalized AuNPs coupled with metformin for the treatment of liver cancer. HA fuctionalized AuNPs were synthesized by reducing HAuCl4using egg-plant (Solanumme longena L.) extract. Metformin-HA coupling was achieved by amide bond formation between amine group of metformin and carboxylic group of HA, activated using EDC and NHS (Fig. 2a). The in vitro cytotoxicity by MTT assay showed that the dose of AuNPs-metformin conjugate required to achieve IC50 was much lower than free metformin. The conjugate also showed better targeted delivery of metformin to HepG2 cells.21 In an another marked research, Joshi et al. have formulated and evaluated thiol functionalized AuNPs conjugated with chloroquine (an anti-malarial, anti-viral and anti-cancer drug) for the cancer chemotherapy (Fig2b). AuNPs were synthesized using sodium borohydride as reducing agent and 11-mercaptoundecnoic acid as thiol functionalizing ligand. The chloroquine was conjugated using standard carbodiimide coupling chemistry with EDC and NHS. Further, the binding of conjugate with serum albumin (SA) was investigated because of its binding with therapeutic agents and transportation to appropriate site. Moreover SA is abundantly present in blood and control many important physiological functions such as control of osmotic pressure, pH buffering etc. The results of binding study with bovine serum albumin (BSA) showed that there was no significant change is structure of protein. Competitive drug binding assay showed that the conjugate binds to warfarin binding site I of subdomain II of protein. Authors concluded that due to efficient binding of conjugate with BSA may lead to the combinatorial therapy i.e drug and radiation for cancer.22

 



Fig. 2. (a) Schematic representation of formation of (a) formation of metformin loaded hyaluronic acid capped AuNP.21(b) 11-mercaptoundecanoic acid functionalized AuNPs and their conjugation to chloroquine.22

 


Khaing Oo et al. had synthesized and evaluated 5-Amino levulinic acid (5-ALA) conjugated AuNPs for photodynamic therapy of cancer. Fibrosarcoma tumor cells, treated with 5-ALA conjugated AuNPs showed preferential increase of protoporphyrin-IX, yielding higher reactive oxygen species as compared to drug alone. The cytotoxic effect of synthesized nanoparticles was observed to be 50% more than 5-ALA alone. The conjugate also showed selective damage to cancerous cells when co-cultured with dermal fibroblasts.23

 

Methotrexate is an important drug which has been conjugated with the AuNPs, and the conjugate is investigated for anticancer activity. Chen et al. had proposed a MTX-AuNP conjugate for the therapy of lung cancer. Methotrexate was directly bound to AuNP via carboxylic group to form MXT-AuNP conjugate (Fig. 3a). The results of in vitro cytotoxicity study showed the faster and higher accumulation of MTX in tumors cells treated with conjugate as compared to free MTX. The cytotoxic effect on different cancer cell lines was also more for the conjugate as compared to equal dose of free MTX. This was attributed due to the faster and high accumulation of MTX in case of MTX-AuNP complex. Administration of MTX–AuNP  suppressed the tumor growth in a mouse ascites model of Lewis lung carcinoma, whereas an equal dose of free MTX had no antitumor effect.24 In another study, Murawala et al. investigated the efficiency of MTX loaded BSA capped AuNPs (Au-BSA-MTX) in preventing the propagation of breast cancer cell lines (MCF-7) based on MTT and Ki-67 proliferation assays(Fig. 3b). Conjugate showed higher cytotoxicity on MCF-7 cells as compared to an equal dose of free MTX. The improved activity was accredited to the favored uptake of conjugate by MCF-7 cells due to the presence of BSA. Bovine serum albumin is used as a source of nutrient and energy for propagation by MCF-7 cells. Moreover, selective binding of MTX to over expressed folate receptors on MCF-7 cells also contributed to the increased cellular uptake and antitumor activity.25

 

6-Mercaptopurine and its riboside derivatives are one of the most widely used anti-leukemic drugs. Podsiadlo et al. have formulated 6-mercaptopurine-9-β-d-ribofuranoside loaded gold nanoparticles and investigated its anti-leukemic activity against K562 leukemia cells (Fig 3c). The authors had reported about significant enhancement in anti-proliferative effect of conjugate as compared to free drug. The enhanced effect was attributed to the increased intracellular transport followed by the subsequent release of drug in lysosomes. Enhanced activity of drug loaded nanocarriers might be helpful in the reduction of the dose of the drug, improved renal clearance and reduced side effects. The nanoparticles with mercaptopurine also showed excellent stability over 1 year without loss of inhibitory activity.26


 

 

Fig. 3.  Schematic representation of formation of (a) methotrexate conjugated AuNPs.24 (b) methotrexate loaded BSA capped AuNPs.25, and (c) 6-Mercaptopurine stabilized AuNPs.26

 


Paclitaxel is important chemotherapeutic agent exhaustively investigated for conjugation with AuNPs. In a pioneer research, Gibson et al. formulated and characterized paclitaxel- functionalized AuNPs. The authors have only performed the chemical characterization of conjugate (no anticancer activity have been reported).27

 

Hwu et al. formulated and investigated systems based on paclitaxel functionalized at the C-2 position using a phosphodiester linkage. Functionalized paclitaxel was attached to a PEG linker containing a thiol group for attachment to citratecapped AuNPs or maleimide-coated magnetitenano particles.

 

The phosphodiester bond was proposed to be positively sliced by phosphodiesterases in tumor cells. Only magnetite nanoparticles showed any substantial release of paclitaxel in the company of phosphodiesterase, that’s also after 4 days of treatment. But, a useful finding and conclusion of the above research was that the water solubility of both types of the (gold and magnetite) nanoparticles was more as compared to free drug, and useful for the treatment of cancer.28

 

Zhang et al have synthesized AuNP-Oligonucleotide (DNA)-Paclitaxel conjugate to improve the solubility of paclitaxel (PTX) in aqueous fluid. Paclitaxel was activated at C-2 position by esterification and coupled with thiolated fluorescent oligonucleotide, which was immobilized onAuNP (Fig 4). The in vitro cytotoxicity study by MTT assay showed increased cytotoxicity as compared to unconjugated paclitaxel. The conjugate also showed toxicity to free paclitaxel resistant cell lines, which indicated that it can be used to improve the drug transport to resistant cells.29

 

Heo et al. in a study, investigated whether the gold AuNP surface-functionalized with PEG, biotin, PTX and rhodamine B linked beta-cyclodextrin (β-CD) (AuNP-5′) can be useful as a theranostic agent for cancer therapy without the cytotoxic effect on normal

 

cells.PTX, an anti-cancer agent, formed inclusion complexations with β-CD conjugated AuNPs, and effectively released from the AuNP-2′ surface-functionalized with PEG, beta-cyclodextrin (β-CD) and paclitaxel (PTX) using the intracellular glutathione (GSH) level (10 mm). Two types of AuNP-4 surface-functionalized with PEG and rhodamine B linked β-CD and AuNP-5 surface-functionalized PEG, biotin and rhodamine B linked β-CD were used for evaluating their specific interaction on cancer cells such as HeLa, A549 and MG63. These were also tested against normal NIH3T3 cell, determining that the AuNP-5 was more effectively involved with the cancer cells. Confocal laser scanning microscopy (CLSM), fluorescence-activated cell-sorting (FACS) and cell viability analyses showed that the AuNP-5′ plays a significant role in the diagnosis and therapy of the cancer cells, and may be used in theranostic agents.30

 

 

Fig. 4. Schematic representation of formation of AuNP-Oligonucleotide (DNA)-Paclitaxel conjugate.29

 

Doxorubicin is another example of drug which is conjugated with the AuNP. Wang et.al has synthesized and investigated doxorubicin bonded AuNP for the breast cancer therapy.31 Drug was covalently bonded through a thioctic acid-PEG linker to the surface of citrate capped AuNPs through a hydrazone group (Fig 5a). The hydrazone get hydrolysed at reduced pH values in endosomes causing the drug release.32 These prepared nanoparticles showed increased cytotoxicity in drug resistant breast cancer cells, as compared to free drug. Kim et al have conjugated fluorescein or doxorubicin to small sized, positively or negatively charged AuNPs (2 nm diameter) and investigated in tumour cylindroids, an in vitro cancer model. The surface of the AuNPs consisted of thioalkyl tetra (ethylene glycol) lyatedtrimethyl ammonium or thioalkyl tetra (ethylene glycol) lyated carboxylic acid to create cationic or anionic AuNPs, respectively(Fig.5b). Positively charged doxorubicin conjugated AuNPs showed higher tumour cells uptake as compared to negatively charged.33Shirazi et al. has synthesized cyclic peptides (CP) capped AuNPsand studied the cellular delivery of doxorubicin in the presence of nanoparticles. Flow cytometry analysis showed the enhanced cellular uptake and nuclear localization of doxorubicin in human ovarian adenocarcinoma (SK-OV 3) cells after 24 h in the presence of CP-AuNP complex as compared to drug alone.34

 

The research can be further extended to conjugate doxorubicin to cyclic peptide capped AuNPs.


 

 

 

 

Fig. 5. (a) Doxorubicin tethered responsive AuNP.31 (b) Doxorubicin or fluorescein conjugated AuNPs.33

 


Agasti et al. has synthesized and evaluated the anticancerous activity of 5-fluorouracil conjugated AuNPs. The drug (containing thiol or zwitterionicthiol for increased solubility) was bonded on the pentanethiol capped nanoparticles through the ultarviolet (UV) light sensitive ortho- nitrobenzyl group. The nanoparticles when irradiated with UV light (365nm) caused the controlled release of the drug from the particle surface to exert cytotoxic effect. The IC50 value on a per particle basis for the AuNPs was 0.7 μM. No significant cell death was observed in cells treated with only light irradiated AuNPs or only light (Fig. 6).35


 

Fig. 6. Schematic representation of basic structure and delivery of payload to cell under photochemical reaction, of 5-Flurouracil conjugated pentanethiol capped AuNPs.35

 


Dreadenet al. had formulated and studied, tamoxifen covalently coupled to citrate capped AuNPs using thiolated PEG linker. Tamoxifen conjugated AuNPs were found to be 2.7 times more potent in breast cancer than free drug. The high potency was due to the increased cellular uptake of complex as compared to free tamoxifen. The increased cellular uptake was facilitated by estrogen receptors, which are over expressed in breast cancer. So, it could be an effective approach for drug targeting in breast cancer.36Another anticancer drug which has been conjugated to the AuNP is Khalalide F. It is polypeptide drug synthesized by cysteine modification and could bind covalently to citrate capped AuNPs. In the study it was observed that the drug loading on AuNP was higher than the expectations from covalent binding. It was attributed to the multilayer coating drug molecules on nanoparticles surface. The drug-nanoparticle conjugate system showed increased anticancer activity due high cellular uptake as compared to free drug. In this work, it was not clear if total amount of drug in the conjugate system is covalently bound or any free drug is present, which makes it difficult to exactly assess the anti-cancerous activity.37Kim et al. designed and fabricated AuNPs, where drug is incorporated in the monolayers on the surface of functionalized nanoparticles (Fig 7). The biocompatible AuNP was having two functional domains, hydrophobic alkanethiol interior and a tetra(ethylene glycol) (TEG) hydrophilic shell, terminated with a zwitterionic head group. Particles having zwitterionic group have shown to minimize the non-specific binding to biological macromolecules. Three different types of hydrophobic guestmoieties: Bodipy, tamoxifen and β-lapachone as drugs were loaded to AuNP. Fluorescence microscopy,

 

using a hydrophobic fluorophore (bodipy), and drug efficacy measurements of therapeutics concluded membrane-mediated diffusion into cells without cellular uptake of the particles. These DDS are great candidates for passive targeting utilizing the enhanced permeability and retention effect.38


 

 

Fig. 7. Schematic representation basic structure of biodipy or tamoxifen or β-lapachone entrapped, surface functionalized AuNPs and delivery of payloads to cancerous cells.38


Cheng et al. has reported the synthesis and evaluation of PEGylated AuNPs-Silicon phthalocyanine 4 (Pc4) conjugate. Silicon phthalocyanine 4 (Pc4) is a hydrophobic photo dynamic therapy (PDT) drug which is under phase I clinical trial for cancer therapy. In in vivo PDT, with AuNP-Pc4 conjugate system the time to achieve maximum drug concentration in the tumor cells reduced significantly to less than 2 h, as compared to 1 or 2 days for free Pc4.  Once Pc4 was delivered and released from AuNPs, the singlet oxygen generation was same as for free Pc4. Thus, AuNP-Pc4 acts as a highly efficient vector for drug delivery in PDT.39Dixit et al. has also formulated PEGylated AuNPs conjugated with Pc4 and investigated, in vitro for the treatment of brain tumors. Cell uptake studies in glioma cell lines, LN229 and U87, showed a significant increase in cellular uptake and intracellular localization for double targeted conjugates as compared to either single targeted Au NPs. Hydrodynamic diameter of these conjugates was found to be within the limits needed to cross the blood brain barrier. So, the efficiency of conjugate can be investigated in vivo using suitable animal models.40 Tomuleasa et al. have synthesized and investigated the antitumor activity of the AuNPs non-covalently conjugated with conventional chemotherapeutic agentsi.ecispaltin, doxorubicin and capecitabine. The AuNPs were stabilized with a monolayer of L-aspartate and cytostatic drugs were non-covalently conjugated to the hydrophilic nanoparticle. The results of in vitro cytotoxicity on cancer cell lines and normal liver cells showed significant decrease in the rate of proliferation of cancerous cells in the presence of AuNP-Drugs conjugate as compared to free drugs.41

 

Anti-bacterial:

Antibiotics/non-antibiotic chemical derivatives conjugated AuNPs have been widely investigated for bactericidal action.

 

Antibiotics-gold nanoparticles conjugates as anti-bacterial:

Several antibiotics have been conjugated either covalently or non-covalently with AuNPs for improved bactericidal property. The several reports published in the reputed journals suggest that mostly antibiotics are conjugated to AuNPs by two ways; amine-gold conjugate and thiol-gold conjugate (Table 2).


 

Table 2. Gold nanoparticles covalently conjugated with antibiotics/chemical derivatives

Antibiotics/Chemical derivatives

Linking agent between AuNP and antibiotic/chemical derivatives

Attachment

Reference

Streptomycin Kanamycin Neomycin Gentamicin

Bovine serum albumin

Amide

46

Streptomycin Kanamycin

-

Amide

44

Cefaclor

-

Amide

47

Ciprofloxacin

-

Amide

48

Acridine derivatives viz. 9-aminoacridine hydrochloride and acridine orange

-

Amide

51

Vancomycin

-

Thiol

52, 53

Toluidine blue O

Tiopronin

Thiol

54

 


Amine bonded antibiotics:

Antibiotics containing free amino groups have been non-covalently conjugated with the gold nanoparticles and investigated for bactericidal property. The AuNPs were conjugated to antibiotics either by simple mixing of stabilized nanoparticle with antibiotic drug or by reduction of gold chloride in presence of antibiotic.42-44 Some reports suggests that the free amino group shows strong affinity towards gold surface, which leads to the aggregation of particles, and conjugate does not show any significant enhancement in activity compared to free drug.45 The problem of stabilization of nanoparticles increased in case of antibiotics containing many amino groups such as aminoglycosides antibiotics. Rastogi et al. have synthesized bovine serum albumin capped AuNPs and conjugated the nanoparticles with aminoglycosides antibiotics viz. streptomycin, neomycin, kanamycin and gentamicin. The drug loaded nanoparticle solutions were evaluated for their antibacterial activity against Gram positive and Gram negative strains. The antibiotic conjugated AuNPs showed enhanced antibacterial activity than antibiotic alone at the same dose.46 Saha et al. have evaluated the antibacterial efficiency of antibiotics viz. ampicillin, streptomycin and kanamycin individually conjugated with AuNPs. Antibiotics were conjugated with AuNPs during synthesis of nanoparticles by utilizing the combined reducing property of antibiotics and sodium borhydride. The antibacterial efficacy of conjugates was evaluated on three different bacterial strains viz. E.Coli, M. luteus and S. aureus. The AuNPs conjugates of streptomycin and kanamycin exhibited enhanced antibacterial activity as compared to free antibiotic. The AuNP conjugate of ampicillin showed decreased activity as compared to free drug. The variation in the activity of the antibiotic conjugates was attributed to the variable binding affinity to the enzymes responsible for catalyzing mechanism of actions of antibiotics.44Rai et al. have used cefaclor, a second generation antibiotic both as reducing and capping agent for the synthesis of AuNP, leaving the β-lactam ring of cefaclor for antimicrobial activity. The conjugate showed enhanced antimicrobial activity against both Gram positive and Gram negative bacteria as compared to antibiotic or AuNPs alone.47Tom et al have conjugated ciprofloxacin to citrate capped AuNPs and suggested as useful carrier for the antibiotic.48

 

Acridine based drugs are well reported for antifungal, antibacterial and antimalarial activity.49, 50 Mittra et al. have conjugated acridine derivatives viz. 9-aminoacridine hydrochloride hydrate (9AA-HCl), acridine yellow (AY), acridine orange (AO) and proflavin (Pro) on citrate stabilized AuNPs. Among the four, only 9AA-HCl and AO showed antibacterial activity. The above two drugs when individually conjugated with AuNPs, the conjugate showed enhanced antibacterial activity compared to free drugs.51

 

Thiol bonded antibiotics:

Standard gold thiol binding method is an important approach for conjugating antibiotics or antibacterial agents to AuNPs. In a pioneering work, Gu et al have conjugated the vancomycin to AuNPs through a phase transfer technique. An aqueous solution of vancomycin was mixed with the solution of quaternary ammonium bromide coated AuNps in toluene, leading to the transfer of coated nanoparticles to aqueous phase. The conjugate was investigated against vancomycin resistant enterococci and some gram negative bacterias viz. E. Coliand showed admirable results even when vancomycin alone was not effective.52 In another research, Huang et al investigated vancomycin bound AuNPsas the photothermal agents for retarding pathogenic bacterial growth, under irradiation of near infrared radiation. Vancomycin conjugated AuNPs showed selective-binding onto the cell walls of bacteria and once bound exhibited high photothermal destruction efficiency of pathogens.53Thetoluidine blue O (TBO), a light activated antimicrobial agent was covalently linked to the carboxyl group on tiopronin-functionalized AuNPs. These conjugates showed four time decrease in minimum bactericidal concentration under white light or laser illumination as compared to free TBO, due to an enhanced light absorbance of AuNPs conjugated with TBO.54

 

Non-antibiotic drug-gold nanoparticles conjugate as anti-bacterial:

There are drugs having no anti-bacterial action alone, but when conjugated with AuNPs showed efficiency against multi drug resistant (MDR) bacterial strains. In a marked research, Zhao e.al reported a new approach of designing antibacterial agents. Amino-substituted pyrimidinthiol (PDT) (themselves completely inactive as antibiotics), when conjugated on AuNPs, show antibacterial properties against multidrug-resistant clinical isolates, without external sources of energy such as IR (Fig 8a). These conjugates were more effective against MDR gram negative strains as compared to gram positive. These conjugates wield their antibiotic activity via sequestration of magnesium or calcium ions to disrupt the bacterial cell membrane, causing leakage of cytoplasmic contents including nucleic acids. These conjugates showed bacterial resistance more slowly compared with conventional, small-molecule antibiotics and appeared harmless to human cells.55 On the basis of findings of above research, Zhao et al in another research conjugated non-antibiotic drugs(NADs) and PDT on AuNPs for synergestic and broad spectrum antibacterial action against MDR superbugs. Several NADs viz. guanidine, metformin, chloroquine, 1,3 (chlorophenyl) biguanide, acetylcholine chloride and melamine were conjugated alone or along with PDT to AuNPs (Fig 8b). The results of antibacterial study showed that the conjugations of NAD and PDT, both on AuNPs showed synergestic and improved activity against MDR gram negative strains. As reported earlier, AuNP-PDT conjugate was less active against gram positive strains, but AuNP-PDT-NAD conjugate demonstrated improved activity against MDR gram positive strains. Conjugate of metformin-PDT-AuNPs, showed the best enhanced activity via increasing the ability to compromise bacterial cell walls. Synergistic effects are also reflected in the eradicating biofilm cells. The findings suggest a large chemical space to develop new antibacterial materials to treat superbugs.56

 

Miscellaneous applications of gold nanoparticles-drug conjugate:

Anti-retroviral agent:

Gold nanoparticle-drug conjugate have been investigated as an anti HIV agent. Bowman et al have synthesized SDC-1721, a fragment of potent anti HIV agent TAK-779 and conjugated it with AuNPs. The anti HIV activity of conjugate was comparable to the Tak-779. SDC-1721 alone does not exhibit any activity in HIV infection. Gold nanoparticle transforms biologically inactive SDC-1721 into a multivalent conjugates that effectively inhibited HIV-1 fusion to human T-lymphocytes.57

 

Anti-arthritic agent:

Gomes et al have developed a MTX-AuNPs conjugated nanoparticle system. The AuNPs were formulated by Turkevich and Frens method and methotrexate was conjugated to nanoparticles by absorption method. The anti-arthritic property of conjugated was investigated on animal model by studying urinary and serum parameters viz. hydroxyproline, glucosamine, pyridoline, deoxypyridoline and serum cytokines. As per the results, conjugate showed significant anti-arthritic property as compared to MTX alone.58


 

Fig. 8. (a) Pyrimidine thiol capped AuNP.55(b) Structures of NADs and pyrimidine thiol; and conjugation of NAD and Pyrimidine thiol to AuNP for synergestic anti-bacterial action.56

 


CONCLUSION:

The drug delivery using AuNPs is an attractive and fastly expanding area of pharmaceutical research. The AuNPs alone have been widely investigated as an anti-cancer, anti-bacterial, anti-arthritic and anti-diabetic agent. The several important and widely used small molecule drugs have been conjugated to AuNPs either covalently or non-covalently for anti-cancer, bacterial infection, HIV and arthritis treatment. The in vitro studies of conjugates have showed improved treatment effect with low dose and fewer side effects as compared to free drug. In future, the design of suitable in vivo models to study the fate of conjugates in animals or humans can be an attractive area for research and, it has already been started in some cases.

 

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Received on 17.02.2018                                   Accepted on 15.03.2018                                                                        

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2018; 8 (1):47-51.

DOI: 10.5958/2231-5713.2018.00008.9