Nanopartikel Emas Multifungsi untuk Aplikasi Diagnostik dan Terapi yang Lebih Baik:Tinjauan

Aplikasi Biologis AuNP

Peran dan signifikansi AuNPs dalam ilmu kedokteran tidak diragukan lagi menjadi lebih terlihat, yang didukung oleh meningkatnya jumlah penelitian yang menunjukkan aplikasi multifaset mereka dalam berbagai bidang biomedis. Biokompatibilitas AuNPs dikaitkan dengan sejarah panjang emas dalam pengobatan penyakit manusia, yang kembali ke 2500-2600 SM. Orang Cina dan India menggunakan emas untuk pengobatan impotensi pria, epilepsi, sifilis, penyakit rematik dan TBC. Cina menemukan efek umur panjang dari koloid emas merah, yang masih dipraktekkan di India sebagai bagian dari pengobatan Ayurveda untuk peremajaan dan revitalisasi. Cinnabar-emas (juga dikenal sebagai Makaradhwaja) digunakan untuk meningkatkan kesuburan di India. Di negara-negara Barat, emas telah digunakan untuk mengobati gangguan saraf dan epilepsi. Tidak ada toksisitas yang dilaporkan untuk penggunaannya dalam studi in vitro dan in vivo [8, 44, 45]. Sejak itu, senyawa emas oral dan injeksi terus digunakan sebagai pengobatan untuk arthritis [9, 46] dan juga telah terbukti memiliki efek antikanker [8]. Efek serupa dan dalam beberapa kasus juga dilaporkan untuk AuNPs, yang muncul sebagai agen yang menjanjikan untuk diagnosis penyakit [47,48,49] dan terapi [3, 29, 50, 51].

AuNPs memiliki luas permukaan yang lebih besar yang dapat dimanfaatkan untuk aplikasi biomedis, dengan menempelkan berbagai biomolekul agar sesuai dengan fungsi yang diinginkan. Ini dapat mencakup bagian penargetan untuk membantu mengenali biomarker spesifik penyakit, agen kontras untuk bio-imaging dan agen terapeutik untuk pengobatan penyakit [24, 25]. Keuntungan menggunakan AuNPs dibandingkan nanomaterial lainnya adalah bahwa mereka dapat dengan mudah difungsikan menggunakan berbagai kimia seperti yang ditunjukkan pada Gambar. 2 [4, 26]. AuNPs memiliki afinitas tinggi untuk molekul tiolasi, dan pengikatan tiol-emas adalah metode yang paling umum digunakan untuk mengadsorbsi molekul ke permukaan NP [4]. Kimia berbasis afinitas seperti pengikatan biotin-streptavidin dan kopling karbodiimida juga digunakan. AuNPs digunakan dalam tiga bidang utama biomedis:pengiriman obat-obatan, tujuan diagnostik dan terapeutik [24, 35], dan telah menunjukkan potensi besar di bidang ini seperti yang dibahas di bawah ini.

Sintesis dan fungsionalisasi AuNPs. Biomolekul dengan gugus fungsi pertama diadsorpsi pada permukaan NP melalui afinitas emas-tiol. Kemudian, gugus fungsi lain seperti gugus amina dapat digunakan untuk mengikat molekul dengan gugus karboksil untuk melampirkan bagian penargetan atau obat. Diadaptasi dari [32]

AuNPs sebagai Agen Pengiriman Obat

Aplikasi AuNPs yang paling umum adalah sebagai pembawa pengiriman obat [11, 18, 52], vaksin [53] dan terapi gen [24, 32]. AuNPs memiliki sifat yang dapat menyelesaikan sebagian besar masalah yang terkait dengan terapi konvensional seperti resistensi obat, distribusi obat yang rendah, biodegradasi dan pembersihan obat awal [11]. AuNPs secara signifikan dapat mengurangi dosis obat, frekuensi pengobatan dan mampu mengangkut obat hidrofobik dan tidak larut. Mereka dianggap bio-inert dan dapat menutupi muatannya dari serangan sel-sel kekebalan, melindungi obat dari degradasi proteolitik saat mereka melakukan perjalanan melalui sistem peredaran darah, dan dengan demikian meningkatkan waktu sirkulasi obat. Faktor-faktor ini dapat dengan mudah meningkatkan kemanjuran obat dengan memusatkan dan mempertahankannya di jaringan yang sakit dengan sedikit atau tanpa efek pada jaringan normal [25].

Penggunaan AuNPs dalam pengobatan kanker telah dipelajari secara ekstensif [17, 37, 54], dan selama bertahun-tahun telah diperluas ke penyakit lain seperti obesitas [50, 55, 56] dan jerawat [18]. Sistem berbasis nano lebih kecil dari sebagian besar komponen seluler dan dapat secara pasif melintasi penghalang seluler dengan memanfaatkan efek peningkatan permeabilitas dan retensi (EPR) pada pembuluh darah jaringan yang sakit [25]. EPR dalam keadaan patologis ditandai dengan angiogenesis yang berlebihan dan peningkatan sekresi mediator permeabilitas, yang dapat meningkatkan penyerapan AuNP oleh jaringan yang sakit. Karakteristik ini hanya terkait dengan keadaan patologis dan bukan jaringan normal, yang memberikan peluang untuk penargetan selektif konjugat AuNP [25]. AuNPs menarik sebagai pembawa obat karena mereka dapat membawa banyak molekul secara bersamaan, semakin mendiversifikasi sifat mereka. Ini adalah sifat yang diinginkan dalam kedokteran di mana sebagian besar aplikasi bio AuNP berakar, karena AuNP dapat disesuaikan untuk fungsi biomedis tertentu. Ini dapat membantu mengontrol cara mereka berinteraksi dengan organel seluler dan oleh karena itu menjanjikan pengembangan masa depan modalitas diagnostik dan pengobatan yang efektif untuk berbagai penyakit [4].

Sistem Diagnostik Berbasis AuNP

Munculnya nanoteknologi telah meningkatkan pendirian dalam mengembangkan sistem deteksi yang cepat, kuat, sensitif dan sangat kompetitif dibandingkan dengan tes diagnostik konvensional [48]. Nanomaterials biasanya terintegrasi dalam platform biosensing yang ada untuk mendeteksi gas, DNA dan penanda protein yang terlibat dalam perkembangan penyakit [47]. Di antara berbagai nanomaterial (yang meliputi NP logam, polimer, magnetik dan semikonduktor) yang digunakan dalam diagnostik, AuNPs telah banyak digunakan dalam biosensor, sensor elektrokimia dan uji kromogenik untuk mendeteksi atau merasakan keberadaan biomarker penyakit [49]. SPR lokal mereka (LSPR), transfer energi resonansi fluoresensi (FRET), hamburan Raman yang ditingkatkan permukaan, konduktivitas, aktivitas redoks, dan efek pengisian terkuantisasi menjadikannya alat yang ideal untuk pencitraan dan deteksi molekul target [10, 24]. Sifat elektronik dan optiknya, dan kemampuan untuk menyebarkan cahaya tampak dan inframerah dekat (NIR) kompatibel dan terukur dengan berbagai teknologi seperti teknik mikroskopis (elektron, confocal, dan hamburan cahaya medan gelap) [57], computed tomography (CT) , Teknik pencitraan PT heterodyne, spektroskopi UV-Vis dan Raman [24, 35].

Pengembangan sistem diagnostik berbasis AuNP melibatkan modifikasi permukaan AuNP, misalnya, melalui perlekatan biomolekul yang mengenali biomarker penyakit [3, 24, 58]. Lateral flow assays (LFA) mungkin merupakan contoh paling terkenal dari alat diagnostik berbasis nanoteknologi. LFA biasanya menggunakan AuNP sekitar 30-40 nm karena partikel yang lebih kecil memiliki penampang kepunahan yang sangat kecil, sedangkan partikel yang lebih besar biasanya tidak stabil untuk digunakan dalam pengujian ini [59]. Selain itu, molekul/enzim lain yang dapat memicu perubahan SPR, konduktivitas, dan redoks AuNP juga disertakan. Indikator-indikator ini memberikan sinyal yang dapat dideteksi setelah pengikatan analit ke konjugat AuNP [24], kekurangan atau adanya sinyal kemudian akan mencerminkan tidak adanya atau adanya molekul target atau penyakit. Sinyal yang dihasilkan oleh AuNPs secara kimiawi stabil, tahan lama dan konsisten ketika digunakan dalam format uji yang berbeda:tabung reaksi, strip, in vitro dan in vivo [24]. Oleh karena itu, penerapannya telah sangat meningkatkan kecepatan dan keberhasilan uji diagnostik.

Uji Berbasis AuNP Kolorimetri

Dalam uji kolorimetri, AuNP menghasilkan sinyal visual (biasanya perubahan warna) yang dapat dideteksi dengan mata telanjang tanpa menggunakan instrumen canggih. Umumnya, larutan koloid AuNPs memiliki warna merah ruby ​​hingga warna anggur yang sangat bergantung pada jarak antar partikel [60, 61]. Pengikatan analit ke AuNPs yang dimodifikasi dengan elemen bio-recognition molekuler (misalnya, antibodi, peptida, aptamers, enzim, dll.) menginduksi pergeseran yang berbeda dalam LSPR, akibatnya menghasilkan perubahan warna dari merah ruby ​​menjadi biru [60 , 62, 63]. Intensitas warna berbanding lurus dengan konsentrasi analit dan digunakan untuk mengkonfirmasi keberadaan dan keadaan penyakit. Diagnostik kolorimetri berbasis AuNP telah berhasil digunakan dalam mendeteksi virus influenza A [64], virus Zika [65], Bakteriofag T7 [66], Mycobacterium tuberculosis [67], dan baru-baru ini, untuk mendeteksi sindrom pernapasan akut parah-coronavirus-2 (SARS-CoV-2) [60, 68].

Contoh uji kolorimetri berbasis AuNP ditunjukkan untuk mendeteksi SARS-CoV-2 [60], virus yang menyebabkan penyakit virus Corona 2019 (COVID-19) yang sangat menular [60, 68]. Dengan pengujian ini, keberadaan virus dilaporkan dengan perubahan warna yang sederhana; tidak ada instrumentasi yang diperlukan untuk melakukan diagnosis. Tes diagnostik klinis virus ini saat ini menggunakan uji reverse transcriptase real-time polymerase chain reaction (RT-PCR), yang memakan waktu 4–6 jam, sedangkan sistem rapid point-of-care (PoC) mendeteksi antibodi yang mungkin memerlukan waktu 4–6 jam. beberapa hari untuk muncul dalam darah. Sebagai perbandingan, uji berbasis AuNP kolorimetri lebih kuat dan lebih cepat seperti yang ditunjukkan pada Gambar. 3. Menginkubasi tag AuNP dengan antisense oligonucleotides (ASO) dengan adanya sampel RNA SARS-CoV-2 menghasilkan pembentukan endapan biru di dalam ~ 10 mnt. Dalam tes positif-SARS-CoV-2, pengikatan ASO ke gen-N dalam fosfoprotein nukleokapsid virus menginduksi warna biru yang terdeteksi secara visual. Tes ini sangat sensitif dan memiliki batas deteksi 0,18 ng/μL untuk RNA SARS-CoV-2 [60].

Sistem diagnostik kolorimetri berbasis AuNP. Deteksi mata telanjang selektif RNA SARS-CoV-2 oleh AuNPs yang dibatasi ASO. Direproduksi dengan izin [60]. Hak Cipta 2020, ACS Nano

LFA berbasis AuNP mengikuti prinsip yang sama seperti yang ditunjukkan pada Gambar 3; namun, alih-alih perubahan warna dalam larutan, garis yang terlihat terbentuk pada strip uji ketika ada analit. Di hadapan analit, AuNPs ditangkap pada garis uji dan membentuk garis merah yang berbeda, yang divisualisasikan dengan mata telanjang. Intensitas garis ditentukan oleh jumlah AuNP yang teradsorpsi [69]. Contoh LFA berbasis AuNP yang sederhana dan cepat ditunjukkan pada Gambar. 4, untuk mendeteksi Pneumocystis jirovecii (P.jirovecii ) antibodi IgM dalam serum manusia. AuNP 40 nm dikonjugasikan dengan antigen sintetik rekombinan (RSA) dari P. jirovecii , baik glikoprotein permukaan utama atau protease serin mirip kexin, yang digunakan sebagai indikator untuk ada tidaknya P. jirovecii . Dalam tes positif, P. jirovecii IgM ditangkap oleh konjugat AuNP-RSA pada bantalan konjugat. Kompleks AuNP-RSA/IgM kemudian mengalir ke membran analitik di mana ia mengikat IgM anti-manusia (garis uji) dan kelebihannya berpindah ke antibodi anti-RSA (garis kontrol), menghasilkan dua garis merah. Tes negatif hanya akan memiliki warna merah pada garis kontrol [70]. Sebuah studi independen menggunakan LFA berbasis AuNP untuk secara selektif mendeteksi IgM SARS-CoV-2 sebagaimana dikonfirmasi oleh munculnya garis merah di kedua garis uji dan kontrol [68]. Warna dideteksi secara visual dengan mata telanjang dalam waktu 15 menit di kedua sistem, dan hanya 10–20 μL sampel serum yang diperlukan per pengujian [68, 70].

LFA berbasis AuNP untuk mendeteksi IgM P. jirovecii antibodi. Ada (tes positif) atau tidak adanya (kontrol negatif) dari P. jirovecii antibodi dapat dibedakan dengan warna kemerahan AuNP di kedua jalur uji dan kontrol, atau hanya di jalur kontrol, masing-masing. Direproduksi dengan izin [70]. Hak Cipta 2019, Perbatasan dalam Mikrobiologi

Salah satu contoh pertama penggunaan AuNPs sebagai probe pensinyalan pada LFA adalah untuk mendeteksi sel Ramos; aptamer TE02 digunakan sebagai probe penangkapan dan aptamer TD05 sebagai probe deteksi. Biosensor aptamer-AuNP dapat secara visual mendeteksi minimal 4.000 sel Ramos tanpa instrumentasi apa pun dan 800 sel Ramos dengan pembaca strip portabel dalam waktu 15 menit. Menggunakan biosensor deteksi sandwich ini, pengujian berhasil mendeteksi sel Ramos yang dibubuhi darah manusia [71] dan digunakan sebagai bukti konsep untuk mengembangkan sistem yang cepat, sensitif, dan berbiaya rendah untuk deteksi kualitatif dan kuantitatif sel kanker yang bersirkulasi. Sejak itu, berbagai LFA berbasis AuNP telah dirancang untuk diagnosis berbagai penyakit menular, termasuk penyakit yang disebabkan oleh Pneumocystis pneumonia [70], virus Ebola [72], HIV, virus Hepatitis C, dan Mycobacterium tuberculosis [73] dan baru-baru ini virus SARS-CoV-2 [68].

Sistem Pencitraan Berbasis AuNP

AuNPs telah diselidiki secara intensif untuk aplikasi dalam bio-imaging karena kemampuannya untuk menyerap dan menyebarkan cahaya yang sesuai dengan panjang gelombang resonansinya, hingga 10 ⁵ kali lebih dari fluorophores konvensional [74]. AuNP memiliki nomor atom dan kerapatan elektron yang lebih tinggi (79 dan 19,32 g/cm ³ ) dibandingkan dengan agen berbasis yodium konvensional (53 dan 4,9 g/cm ³ ), sehingga terbukti menjadi agen kontras yang lebih baik [24]. AuNPs menumpuk pada sel atau jaringan yang sakit dan menginduksi redaman sinar-X yang kuat membuat situs yang ditargetkan sangat berbeda dan mudah dideteksi. AuNPs melekat pada bagian kimia dan agen bio-rekognisi molekuler yang secara selektif dapat menargetkan antigen spesifik untuk menginduksi kontras yang berbeda dan target spesifik untuk pencitraan CT [75].

Sistem pencitraan CT molekuler yang ditargetkan secara in vitro dicapai dengan menggunakan AuNP yang difungsikan dengan RNA aptamer yang mengikat antigen membran spesifik prostat (PSMA). Konjugat aptamer AuNP-PSMA menunjukkan intensitas CT lebih dari empat kali lipat untuk sel prostat yang mengekspresikan PSMA (LNCaP) dibandingkan dengan sel prostat PC-3, yang tidak memiliki reseptor target [76]. Demikian pula, konjugat aptamer AuNP-diatrizoic acid-AS1411 dilokalisasi dalam sel CL1-5 (adenokarsinoma paru-paru manusia) dan tikus pembawa tumor CL1-5. Aptamer AS1411 menargetkan reseptor nukleolin (NCL) yang diekspresikan oleh sel CL1-5 pada permukaan sel, sedangkan asam diatrizoat adalah zat kontras berbasis yodium. Konjugat aptamer AuNP–diatrizoic acid–AS1411 memiliki kurva atenuasi linier dengan kemiringan 0,027 mM Au Hounsfield unit (HU ⁻¹ ) menunjukkan akumulasi AuNPs di lokasi tumor [77]. AuNPs menunjukkan waktu retensi vaskular yang lebih lama, yang memperpanjang waktu sirkulasi mereka dalam darah [77,78,79] dan meningkatkan sinyal CT asam diatrizoat [77].

Gambar 5 menunjukkan pencitraan vaskular CT in vivo arteri koroner menggunakan AuNPs yang terkonjugasi ke protein adhesi pengikat kolagen 35 (CNA35) untuk menargetkan kolagen I pada infark miokard pada hewan pengerat. Sinyal AuNP masih terdeteksi dalam darah 6 jam setelah pemberian intravena (i.v), yang secara signifikan lebih tinggi daripada waktu paruh (5-10 menit) agen berbasis yodium [79]. Efek-efek ini direplikasi dengan menggunakan AuNPs mannan-capped yang disintesis hijau, yang menunjukkan serapan yang dimediasi reseptor dan non-toksisitas dalam sel yang mengekspresikan mannose (DC 2.4 dan RAW 264.7). AuNPs mannan-capped selektif menargetkan kelenjar getah bening poplitea in vivo setelah injeksi ke kaki belakang tikus [38]. Pencitraan CT berbasis AuNP dapat memberikan informasi yang signifikan untuk diagnosis berbagai penyakit tidak terbatas pada arteri koroner dan kanker [76, 77, 79,80,81]. Penggunaan AuNPs sebagai agen kontras telah menunjukkan potensi dalam sistem pencitraan lain seperti fotoakustik, pencitraan nuklir, ultrasound dan pencitraan resonansi magnetik. Sistem ini ditinjau secara ekstensif di tempat lain [82, 83].

Pencitraan CT in vivo menggunakan AuNPs sebagai agen kontras CT. AuNPs Mannan-capped dan pencitraan CT mereka dari kelenjar getah bening (A ), dan pencitraan CT AuNPs terkonjugasi CNA35 dari beban parut miokard (B ). Direproduksi dengan izin [79]. Hak Cipta 2018, Elsevier

AuNP dalam Sistem Deteksi Berbasis Fluorescent

AuNP digunakan dalam sistem deteksi berbasis fluoresen sebagai agen fluoresen atau pemadam fluoresen. Pada ukuran ≤ 5 nm, AuNP menampilkan properti titik kuantum (QD) dan dapat digunakan sebagai gantinya. Au₅₅ (PPh₃ )₁₂ Kl₆ nanocluster diperkenalkan pada tahun 1981 mungkin yang paling intensif dipelajari karena perilaku ukuran kuantum mereka [7]. Sejak itu, berbagai AuNPs berukuran kuantum (AuNPsQ) seperti Au₂₅ (SR)₁₈ , Au₃₈ (SR)₂₄ dan Au₁₄₄ (SR)₆₀ [84] telah dipelajari sebagian besar dalam penginderaan elektrokimia karena mereka adalah konduktor elektronik yang sangat baik dan mediator redoks [85].

Elektroda film AuNPsQ digunakan dalam pembuatan imunosensor elektrokimia ultrasensitif untuk mendeteksi antigen spesifik prostat (PSA). Imunosensor memiliki sensitivitas 31,5 A mL/ng dan batas deteksi 0,5 pg/mL untuk PSA dalam 10 L serum manusia yang tidak diencerkan. Immunoassay berkinerja delapan kali lipat lebih baik daripada imunosensor hutan karbon nanotube yang dilaporkan sebelumnya yang mengandung banyak bagian, pada konsentrasi biomarker yang lebih rendah daripada tingkat yang terkait dengan keberadaan kanker. Dengan demikian, dapat digunakan untuk mengukur biomarker uji baik dalam keadaan normal maupun sakit. Kinerja imunosensor sebanding dengan metode ELISA referensi [86]. AuNPsQ juga dimasukkan ke dalam CaCO yang berstruktur berpori₃ bola untuk membentuk CaCO fluoresen₃ /AuNPsQ hybrid untuk mendeteksi enolase spesifik neuron, biomarker diagnostik dan prognostik untuk cedera otak traumatis dan kanker paru-paru. Sensor memiliki batas deteksi 2.0 pg mL ⁻¹ [87]. Sampai saat ini, beberapa sistem deteksi fluoresen berbasis AuNP telah dilaporkan untuk mendeteksi analit yang terkait dengan Hepatitis B [73, 88], Influenza A [89], kanker [90] dan cedera jantung [91].

AuNP juga merupakan quencher berbasis FRET yang sangat baik [92]. Sifat optiknya yang unik (intensitas sinyal yang stabil dan ketahanan terhadap photobleaching), ukuran dan kemampuan untuk dimodifikasi telah membuatnya menjadi probe yang menarik dalam platform penginderaan fluoresensi [93, 94]. AuNP yang lebih besar (≥ 10–100 nm) memiliki hasil kuantum rendah yang tidak cocok untuk penginderaan fluoresen langsung; namun, kemampuannya untuk memadamkan pewarna fluoresen di bawah keadaan energi eksitasi yang relatif tinggi telah menjadikannya sebagai pemadam fotoluminesensi yang efektif [94]. Pada prinsipnya, nanoprobe fluoresensi terdiri dari donor fluorofor (pewarna atau QDs) dan akseptor AuNPs, dan ketika dibawa ke dekat, fluoresensi dari fluorofor yang dipilih dipadamkan oleh AuNPs [94, 95]. Dengan tidak adanya target seperti yang ditunjukkan oleh kurangnya sinyal fluoresen, probe asam nukleat berhibridisasi dan membentuk struktur melingkar yang membawa fluorofor dan quencher pada ujung yang berlawanan menjadi dekat; sementara pengikatan analit ke probe asam nukleat menggantikan fluorofor dari AuNPs yang menghasilkan sinyal fluoresen [24, 94, 96]. Mengambil keuntungan dari sifat-sifat yang disebutkan di atas, AuNP digabungkan dalam suar molekuler untuk deteksi in vitro (nanosfer emas, AuNSs) dan in vivo (nanorod emas, AuNRs) dari ekspresi matriptase pada sel tumor. Dua suar molekuler terdiri dari situs pembelahan matriptase sebagai penghubung antara AuNP dan fluorofor. Suar molekuler AuNS dibuat dengan fluorescein isothiocyanate (FITC), dan suar molekuler AuNR memiliki pewarna fluoresen NIR (asam merkaptopropionat, MPA). In the absence of the target, the AuNSs and AuNRs, respectively, blocked the FITC and MPA fluorescence. Cleavage of either FITC or MPA from the AuNP–molecular beacons in the presence of matriptase exhibited a quantifiable fluorescence signal. The fluorescent signal of the MPA–AuNR–beacon in the nude mice bearing HT-29 tumors lasted for 14 h in the tumor site, while the signal gradually disappeared from the non-tumor site over time [97].

The AuNPs were reported to have comparable or higher fluorescence quenching efficiency than organic quenchers such as 4-((4′-(dimethyl-amino)phenyl)azo)benzoic acid (DABCYL) [94, 98] and Black Hole Quencher-2 [99]. The fluorescence quenching efficiency of 1.4 nm AuNPs was compatible with the four commonly used organic fluorophores (FITC, rhodamine, texas red and Cy5). The fluorescence quenching efficiency of the AuNPs was similar to that of DABCYL, and unlike DABCYL, the AuNPs showed consistency in both low and high salt buffers [98]. In a competitive hybridization assay, 10 nm AuNPs showed superior (> 80%) fluorescence quenching efficiency for Cy3 dye than the commercial Black Hole Quencher-2 (~ 50%). The assay had a limit of detection of 3.8 pM and a detection range coverage from 3.8 pM to 10 nM for miRNA-205 in human serum, and it was able to discriminate between miRNAs with variations in their nucleotide sequence [99]. The competitive sensor arrays were not only sensitive [96, 99] but were able to differentiate between normal and diseased cells, as well as benign and metastatic cancers [96].

AuNP-Based Bio-barcoding Assay

AuNP-based bio-barcoding assay (BCA) technology has become one of the highly specific and ultrasensitive methods for detection of target proteins and nucleic acids up to 5 orders of magnitude than the conventional assays [100]. The assay relies on magnetic microparticle probes, which are functionalized with antibodies that bind to a specific target, and AuNP probes encoded with DNA that recognizes the specific protein target and antibodies. Upon interaction with the target DNA, a sandwich complex between the magnetic microparticle and AuNPs probes is formed. The sandwich is then separated by the magnet followed by thermal dehybridization to release the free bar-code DNA, enabling detection and quantification of the target [101, 102].

The AuNP-based BCA assay was able to detect HIV-1 p24 antigen at levels that was 100–150-fold higher than the conventional ELISA [103]. The detection limit of PSA using these systems was 330 fg/mL [104]. The versatility of AuNPs for the development of a BCA-based platform was further demonstrated by measuring the concentration of amyloid-beta-derived diffusible ligands (ADDLs), a potential Alzheimer's disease (AD) marker found in the cerebrospinal fluid (CSF). ADDL concentrations were consistently higher in the CSF taken from the subjects diagnosed with AD than in non-demented age-matched controls [105]. These results indicate that the universal labeling technology can be improved through the use of AuNPs to provide a rapid and sensitive testing platform for laboratory research and clinical diagnosis.

AuNP-Based Therapies

Metal-based drugs are not new to medicine; in fact, they are inspired by the existing metallic drugs used in clinical treatment of various diseases [9, 106,107,108,109]. The widely studied and clinically used metal-based drugs were derived from platinum (e.g., cisplatin, carboplatin, tetraplatin for treatment of advanced cancers), bismuth (for the treatment of infectious and gastrointestinal diseases), gold (for the treatment of arthritis) and gallium (for the treatment of cancer-related hypercalcemia) [108, 109]. The approval of cisplatin in 1978 by the FDA for the clinical treatment of cancer [107] further inspired research on other metals (such as palladium, ruthenium, rhodium) [32, 106, 110].

Owing to the bioactivities, which included anti-rheumatic, antibacterial and anticancer effects, and the biocompatibility of bulk gold [8, 9, 46, 111], AuNPs are extensively investigated for the treatment of several diseases. AuNPs displayed unique and novel properties that are superior to its bulk counterpart. AuNPs are highly stable and have a distinct SPR, which guides their application in medicine [112], as drug delivery and therapeutic agents. AuNPs have a lot of advantages over the conventional therapy; they have a longer shelf-life and can circulate long enough in the system to reach their targets [25] with [11, 49, 113] or without targeting molecules [14, 15, 24, 25, 114]. AuNPs can provide localized and selective therapeutic effects; some of the areas in which AuNPs were used in therapy are described below.

Therapeutic Effects of Untargeted AuNPs

The as-synthesized (i.e., unmodified or uncapped) AuNPs have been shown to have diverse therapeutic effects against a number of infectious [115, 116], metabolic and chronic diseases [3, 29, 50, 51]. Their antioxidant, anticancer, anti-angiogenic [3, 32], anti-inflammatory [3, 51] and weight loss [29, 50, 112] effects are beneficial for diseases such as cancer, rheumatoid arthritis, macular degeneration and obesity [5, 25, 113, 117]. The above-mentioned diseases are characterized by a leaky vasculature and highly vascularized blood vessels [5, 113], which provides the NPs an easy passage into the diseased tissues and increase the susceptibility of cells to their effects. Through the EPR effect, uncapped AuNPs can passively accumulate in the vasculature of diseased cells or tissues. Hence, AuNPs have been specifically designed to have anti-angiogenic effects in diseases where angiogenesis (the growth and extension of blood vessels from pre-existing blood vessels) spins out of control like cancer, rheumatoid arthritis, macular degeneration and obesity [5, 25, 113, 117]. Targeting and destroying the defective blood vessels prevent oxygen and nutrients from reaching the diseased cells, which results in their death. The pores in the blood vessels at the diseased site (especially in cancer and obesity) are 200–400 nm and can allow materials in this size range to pass from the vasculature into the diseased tissues and cells [14, 15, 25, 114].

The cellular uptake, localization, biodistribution, circulation and pharmacokinetics of the uncapped AuNPs rely strongly on size and shape [49]. Although these effects are applicable to all AuNPs, the biological effects of citrate-capped AuNPs (cAuNPs) are extensively studied and reviewed. Spherical cAuNPs demonstrated selective in vitro anticancer activity that was size and concentration dependent on murine and human cell lines [3, 51]. Different sizes (10, 20 and 30 nm) of cAuNPs showed differential effects in human cervical carcinoma (HeLa), murine fibroblasts (NIH3T3) and murine melanoma (B16F10) cells. The 20 and 30 nm cAuNPs showed a significant cell death in HeLa cells starting at the lowest concentration of 2.2 µg/mL, while the 10-nm NPs was toxic at concentrations ≥ 8.75 µg/mL. The activity of these NPs was negligible in the noncancerous NIH3T3 cells, especially the 10 and 20 nm. The 20 nm reduced viability by ≤ 5% at the highest concentration (35 µg/mL), and ~ 20% for the 10 and 30 nm. The IC₅₀ values for 10, 20 and 30 nm cAuNPs in the Hela cells were 35, 2.2 and 4.4 μg/mL, respectively, while the IC₅₀ values for noncancerous cells were higher than 35 µg/mL [3]. Using a concentration range of 0.002–2 nM, 13 nm cAuNPs induced apoptosis in rabbit articular chondrocytes and no effects were observed for 3 and 45 nm cAuNPs under the same conditions. The 13 nm cAuNPs induced mitochondrial damage and increased reactive oxygen species (ROS); these actions could not be blocked by pre-treatment with a ROS scavenger, the N-acetyl cysteine [51]. Size-dependent effects were also observed in vivo after injecting cAuNPs of various sizes (3, 5, 8, 12, 17, 37, 50 and 100 nm) into mice (8 mg/kg/week) for 4 weeks. The 8, 17, 12 and 37 nm were lethal to the mice and resulted in tissue damage and death after 14 days of treatment; the other sizes were not toxic and the mice survived the experimentation period. On the contrary, the same-size AuNPs at a concentrations up to 0.4 mM were not toxic to HeLa cells after 24 h exposure [118].

The cAuNPs can interact and accumulate nonspecifically within various tissues and organs in the body, especially in the reticuloendothelial system (RES) organs (blood, liver, spleen, lungs) [55, 119]. This was evident in high-fat (HF) diet-induced obese Wistar rats [55] and Sprague–Dawley rats [119] following acute (1 dose for 24 h) [55] and chronic (1 dose; 0.9, 9 and 90 µg/week over 7 week period) [119] exposure to 14 nm cAuNPs, respectively. Majority of the i.v injected cAuNPs were detected in the liver, spleen, pancreas, lungs, kidneys [55, 119] including the skeleton and carcass of the rats [119]. Chen et al. observed that after intraperitoneal (i.p) injection of a single dose (7.85 µg/g bodyweight) of 21 nm cAuNPs in lean C57BL/6 mice, they accumulated in the abdominal fat tissues and liver after 24–72 h [29], as well as the spleen, kidney, brain and heart in the HF-induced obese mice that were injected with the same dose daily for 9 weeks [50]. The cAuNPs reduced the abdominal WATs (retroperitoneal and mesenteric) mass and blood glucose levels 72 h post-injection [29]. In the diet-induced obese mice, the 21 nm cAuNPs demonstrated anti-inflammatory and anti-obesity effects [50]. They also improved glucose tolerance, enhanced the expression of inflammatory and metabolic markers in the retroperitoneal WATs and liver [50]. Both the 14 and 21 nm cAuNPs showed no sign of toxicity or changes in the markers associated with kidney and liver damage [29, 55, 119].

Similar findings were reported for plant-mediated AuNPs, without targeting molecules they can access, ablate tumors [40, 120] and obese WATs [121] in rodents. Differential uptake, distribution and activity of biogenic AuNPs also vary depending on the size and shape of the NPs. While certain sizes can pass through the vascular network and be retained at the site of the disease; others can be easily filtered out of the system through the RES organs and the mononuclear phagocytic system as shown in Fig. 6 [15, 114]. NPs can be removed by tissue-resident macrophages (TRMs) before they reach the disease cells. Those that escape the TRMs and do not reach the disease site, especially smaller NPs (≤ 5 nm), are excreted through glomerular filtration in the kidney [25, 114]. Pre-treatment with clodronate liposomes depleted the TRMs in the liver and spleen before exposure to 50, 100 and 200 nm AuNPs. This reduced uptake of the AuNPs by the liver, increased their half-life in the blood as well as their accumulation at the tumor site [122]. However, TRMs are not the only obstacle that the AuNPs that rely on EPR effect for uptake must overcome. EPR effect alone can only ascertain ≤ 1% AuNP uptake [15, 114], and depletion of the TRMs prior to treatment resulted in just ≤ 2% of NPs reaching the target [122]. The success of non-targeted AuNPs depends on their ability to reach and accumulate in the diseased tissues, of which passive targeting through the EPR effect might not be efficient. The NPs also need to circulate longer, escape early clearance, and most importantly show reduced bystander effects [25, 123]. These qualities can increase bioavailability and ensure selectivity and efficacy of the AuNPs. These can further be improved by changing the surface chemistry of the AuNPs as discussed below [15, 124].

RES-based clearance of systemic administered AuNPs depends on their size. Large AuNPs accumulate in the liver, while smaller AuNPs are likely to end up in the spleen or be excreted in the urine via glomerular filtration. The AuNPs that escape the TRMs could accumulate in the diseased tissues. Reproduced with permission [114]. Copyright 2019, Frontiers in Bioengineering and Biotechnology

Therapeutic Effects of Surface-Functionalized AuNPs

The common strategy in AuNP-based therapeutics involves modifying the AuNP surface with therapeutic agents [3, 124,125,126]. The therapeutic agents can be drugs already used for the treatment of a particular disease or biomolecules with known inhibitory effects on cell signaling. In some instances, the therapeutic AuNPs have also been designed to have molecules that facilitate active targeting of the AuNPs toward specific cells and tissues. The molecules can easily adsorb on the AuNP surface by thiolation, chemical modification using chemistries such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), streptavidin/biotin binding [3, 124,125,126,127] and ionic interactions based on opposite charges between the NP surface and the biomolecules [124,125,126]. Functionalization of the AuNP surface influences their physicochemical properties and can affect their safety, biocompatibility and mobility. To ensure that the cargo carried by the AuNPs is delivered to the intended site, consideration should thus be given to both the physical and chemical properties of the AuNPs [124,125,126]. It is especially the size, shape, charge and the capping agents of the AuNPs that play an important role in the functionality of the AuNP conjugates [124] and can completely alter the pharmacokinetics of the AuNP-based therapeutics.

Functionalization allows for the development of customized nanosystems to reduce undesirable bystander effects often associated with traditional medicine. Functionalization of AuNPs can also prevent nonspecific adsorption of proteins onto the AuNP surface which can result in the formation a protein corona, resulting in the early clearance of the AuNPs through opsonization by the phagocytic cells [49, 123]. The surface charge of NPs can have a major influence on the behavior of NPs within biological environments. AuNPs with a neutral surface charge are unreactive and have a higher rate of escaping opsonization than charged AuNPs. Hydrophilic NPs will also behave differently to those with hydrophobic surfaces [49, 123]. PEG is one of the polymers most often used to mask AuNPs from phagocytic cells and has been shown to stabilize and enhance the biocompatibility of the AuNPs in numerous in vivo studies [49, 55, 123]. Pegylation improved the biocompatibility of 8.2 nm AuNPs by preventing neutrally and negatively charged AuNPs to bind to cell membranes or localize to any cellular components in African green monkey kidney (COS-1) cells [49]. And when the pegylated AuNPs were functionalized with a polyarginine cell penetrating moiety, the AuNPs were visualized on the cell membrane and inside the COS-1 cells [49]. Cell-penetrating peptides such as nuclear localization signal from SV40 virus, Tat from HIV and polyarginine peptides have been explored in translocation of AuNPs inside all cell type, normal or diseased. However, high specificity is required for clinical applications and can be achieved by taking advantage of the physiological differences between malignant and normal cells. This has been achieved by functionalizing the AuNPs with targeting molecules that recognize cell-specific receptors that are exclusively or overexpressed on the surface of target cells. This way, the AuNPs can be directed and delivered only to cells that express the target receptor. Therefore, conjugation of targeting moieties to the AuNPs (active targeting) will provide more selectivity, reduced bystander toxicity and enhanced efficacy since the AuNPs will be confined only to malignant tissues that express the target receptors [49, 55, 57, 113, 126, 127].

A good example to demonstrate the versatility of AuNPs is shown in Fig. 7, where four different molecules were conjugated onto the AuNPs to target two independent markers and mechanisms [11]. The multifunctional AuNPs were used for the treatment of leukemia (K562DR) cells that are resistant to doxorubicin (Dox). The 40 nm AuNPs were modified with two targeting moieties (folate and AS1411 aptamer) and two therapeutic agents (Dox and anti-miRNA molecules/anti-221). Folate molecule and AS1411 aptamer, respectively, recognize the folate and NCL receptors that are overexpressed on the cell surface and through receptor-mediated endocytosis will traffic the AuNP-conjugate into the cells. The AS1411 aptamer had dual functions, by also targeting the NCL receptor that is expressed inside the cells. After the AuNP-conjugate has been shuttled into the cells, the cargo (AS1411 aptamer, anti-221 and Dox) is off-loaded which independently act on three mechanisms that will synergistically bring about the demise of the cells. AS1411 aptamer together with anti-221 prevented leukemogenesis by suppressing the endogenous NCL and miR-221 function in the NCL/miR-221 pathway, thereby sensitizing the cells to the effects of Dox [11].

Multifunctional AuNPs in the treatment of multidrug-resistant (MDR) leukemia cells by increasing the sensitivity of the cells to Dox. Reproduced with permission [11]. Copyright 2019. Springer Nature. Folate (FA) receptor

Interestingly, similar dual targeting and treatment effects were achieved with green synthesized AuNPs without any additional molecules. With natural products acting as reducing agents, the biogenic AuNPs might also be more biocompatible than the chemically synthesized NPs [12, 40, 41, 43, 120]. MGF-AuNPs selectively targeted the laminin receptors in prostate (PC-3) and triple-negative breast cancer (MDA-MB-231) cells, and their xenografts in severe combined immunodeficiency (SCID) mice bearing these tumors [12, 40, 120]. In the normal SCID mice, the majority (85% at 30 min increasing to 95% after 24 h) of the i.v-injected MGF-AuNPs accumulated in the liver. Less than 10% were detected in the blood (2.7%), spleen (5%), lungs (0.6%), stomach, intestines and kidneys. When intra-tumorally injected in SCID mice-bearing prostate tumors, only 11% of the MGF-AuNPs were detected in the liver 24 h post-injection, while ~ 80% was in the tumor. Negligible amounts were found in the stomach, carcass and the small intestines. Some of the AuNPs were excreted through the renal and hepatic pathways in the urine and feces after 24 h [40, 120]. Nano Swarna Bhasma, a mixture consisting of AuNPs synthesized from mango peel extracts and phytochemicals from mango, turmeric, gooseberry and gum arabic, showed reduced toxicity toward normal endothelial cells after 48 h compared to the MDA-MB-231 cells [12].

Several studies have demonstrated that AuNPs have potential for clinical application. In combination with conventional drugs, it can be used to sensitize diseased cells to the drug effects [12, 128] and also prevent or reduce drug-related bystander effects [12]. AuNPs improved the pharmacokinetics of chemotherapeutic drugs, such as Dox [43, 129] and 5-fluorouracil (5-FU) [128]. Great improvements were mostly seen in the permeability and retention of drugs in the diseased cells, resulting in enhanced efficacy [130]. Dox-loaded AuNPs, which were non-toxic toward normal mouse fibroblast (L929) cells, also demonstrated selective toxicity toward fibrosarcoma tumors in mice [129]. 5-FU conjugated to the cAuNPs had better activity than 5-FU on its own in colorectal cancer cells [128]. AuNP co-treatment with chemotherapeutic drugs was highly efficient in improving the efficacy of chemotherapeutic drugs [12, 43, 128, 129, 131]. Orally ingested Nano Swarna Bhasma in combination with Dox and Cyclophosphamide reduced tumor volumes in SCID mice-bearing breast tumor cells and also showed acceptable safety profile and reduced bystander effects of the chemotherapeutic drugs in stage IIIA/B metastatic breast cancer patients [12]. Active targeting alone can ensure that the AuNPs are directly delivered into the desired targets, achieving a balance between efficacy and toxicity while minimizing damage to healthy tissues [14, 15, 49]. Controlled drug release is also among the many advantages offered by the AuNP-based systems and is crucial as it allows for localized and selective toxicity [49]. The AuNPs can be designed in such a way that their conjugates respond to internal (glutathione displacement, enzyme cleavable linkers, pH) or external (light, heat) stimuli to function [24, 25, 34, 128].

AuNPs as Transfection Agents in Gene Therapy

The use of AuNPs in gene therapy has shown promising outcomes by facilitating the delivery of genetic material to cells to silence or enhance expression of specific genes [24, 32, 132]. Thus, AuNPs can be used as transfection reagents in gene therapy for the treatment of cancer and other genetic disorders. AuNP conjugates have demonstrated higher transfection efficiency than experimental viral and non-viral gene-delivery vectors including polycationic reagents that has been approved for clinical use [24].

AuNPs are highly conductive and well suited for use as microelectrodes during electroporation for intracellular delivery of biomolecules for disease treatment. AuNPs significantly enhanced the performance of electroporation systems and have been used successfully for the delivery of DNA into hard-to-transfect cells such as the K562 cells [133]. To prevent cell loss which is often associated with electroporation, targeting moieties can be conjugated to the AuNPs to facilitate cellular uptake of AuNP conjugates through receptor-mediated mechanisms [133]. The use of AuNPs to transfect cells with oligonucleotide molecules also has the added advantage of increasing the half-life of these biomolecules and their efficacy [24, 32].

Untargeted AuNP conjugates are passively transported into cells and rely on the surface charge and AuNP shape for efficient transfection [24, 36, 134, 135]. The charge of the biomolecules that are conjugated onto AuNP surface plays a crucial role in their transfection efficiency; for instance, AuNPs functionalized with cationic molecules produce higher transfection efficiency than AuNPs functionalized with anionic molecules. Positively charged amino acids (lysine) can be attached on the NP surface to increase the rate of transfection. AuNSs [24] and AuNRs [36, 134, 135] are commonly used for transfections, and relative to the conventional transfection reagents (X-tremeGENE and siPORT), they inhibited the expression of target gene by > 70% in vitro [134] and in vivo [135]. In these studies, transfection efficiency was quantified based on target expression using RT-PCR and immunostaining [134, 135]. As transfection reagents, AuNPs provide long-lasting effects, localized gene delivery and higher efficacy [36, 134, 135]. Other types of nanomaterials (e.g., polymeric, liposomes, ceramic and carbon nanotubes) had received more attention for use in gene therapy than AuNPs. Six clinical trials using either polymeric or lipid-based nanomaterials for delivery of siRNA in solid tumors have been completed [36, 134, 136]. All of which suffer from low loading efficiency, low stability, and insufficient payload release [36, 136]. On the other hand, transfection systems based on AuNPs make use of easy chemistry that ensures efficient loading capacity and formation of stable complexes [36, 135]. Their safety can be controlled by manipulating their shape, size distribution and surface composition [36].

Antimicrobial Effects of AuNPs

MDR microbes are a major health concern and a leading cause of mortality, worldwide [21, 137,138,139,140,141]. These microorganisms have become resistant to conventional antimicrobial agents, due to over-prescription and misuse of these drugs [142]. No new antibiotics have been produced in over 40 years, mainly because the big pharmaceutical companies have retreated from their antibiotic research programs due to the lack of incentives [143]. As such, new and effective antimicrobial agents are urgently required to combat what could be the next pandemic, the antimicrobial resistance, and avoid surge in drug-resistant infections.

AuNPs are among the new generation of antimicrobial agents under review. They have shown broad antimicrobial (bactericidal, fungicidal and virucidal) effects against a number of pathogenic and MDR microorganisms and thus have potential to overcome microbial drug resistance [21, 142, 144]. Their antimicrobial effects are dependent on their physicochemical properties, especially their size, surface composition, charge and shape [21, 144]. Due to their small size, AuNPs can easily pass through the bacterial cell membrane, disrupt their physiological functions and induce cell death [35]. The exact antimicrobial mechanisms of AuNPs are not yet fully elucidated; despite this, some of the reported modes of actions that results from the interaction of various nanostructured materials (NSMs) with the bacterial cells are illustrated in Fig. 8. The highlighted mechanisms are also implicated in antimicrobial activity of AuNPs, they include induction of microbial death through membrane damage, generation of ROS and oxidative stress, organelle dysfunction, and alteration of gene expression and cell signaling [141].

Antimicrobial mode of actions of the NSMs. Various NSMs can induce cell death by altering various biological functions, X represents alteration of cell signaling by de-phosphorylation of tyrosine residues in proteins as one of the mechanisms. Reproduced with permission [141]. Copyright 2018, Frontiers in Microbiology

AuNPs have multiple roles to play toward the development of antimicrobial agents, aside from being antimicrobial agents by themselves; they can serve as drug sensitizers and drug delivery vehicles [35, 58, 132, 145]. These features are applicable to both the chemical and green synthesized AuNPs, which have been reported to have antimicrobial effects against a number of human [21, 145,146,147] and waterborne [148] pathogenic strains. Generally, the test bacteria had shown low susceptibility toward the chemically synthesized AuNPs, i.e., the cAuNPs [21, 146, 147] and the NaBH₄ -reduced AuNPs [149]. This was due to the repulsive forces between the negative charges on the AuNP surfaces and bacterial cells, thus preventing the interaction between AuNPs and the bacteria [21]. The activity of chemically synthesized AuNPs is based on their size, shape, concentration and exposure time. As an example, one study reported that NaBH₄ -reduced AuNPs had no activity against Staphylococcus aureus (S. aureus ) and Escherichia coli (E. coli ) at 500 µg/mL for the duration of 6 h [149]. In contrast, another study showed a significant dose (1.35, 2.03 and 2.7 μg/mL) and size (6–34 nm vs 20–40 nm) dependent antibacterial effects of the NaBH₄ -reduced AuNPs on Klebsiella pneumonia , E. coli , S. aureus and Bacillus subtilis [145].

The AuNPs are either used alone or in combination with other antimicrobial agents to treat microbial infections [35, 58, 132, 145]. When used in combination with other antimicrobial agents, the AuNP conjugates resulted in synergistic antimicrobial effects that surpassed the individual effects of the AuNPs and drugs [21, 35, 58, 132, 150]. These drugs were conjugated onto the AuNPs by either chemical methods [4, 151] or the drugs were used as reducing and capping agents [21, 149]. By so doing, the AuNPs improved drug delivery, uptake, sensitivity and efficacy. Some of the FDA-approved antibiotics and non-antibiotic drugs that were loaded onto the AuNPs are shown in Table 1 [4, 21, 149, 152]. Ciprofloxacin [152], cefaclor [149], lincomycin [4], kanamycin [21], vancomycin, ampicillin [151] and rifampicin [32] are among the antibiotics loaded on the AuNPs and demonstrated the versatility of AuNPs. These strategies were successful with various sizes and shapes of AuNPs, including gold silica nanoshells [152], AuNP-assembled rosette nanotubes [151] and AuNPs encapsulated in multi-block copolymers [153]. For instance, cefaclor-reduced AuNSs inhibited the growth of S. aureus and E. coli within 2–6 h depending on the concentration (10–50 µg/mL), while complete bacterial growth inhibition by the drug alone was only observed at 50 µg/mL after 6 h. The minimum inhibitory concentration (MIC) of the treatments was 10 µg/mL and 50 µg/mL for cefaclor-AuNPs and cefaclor, respectively [149].

AuNPs have presented properties that make them ideal candidates as alternative antimicrobial agents; the most important being their broad antimicrobial activity [21, 35, 58, 132, 150]. Owing to their biocompatibility and easily modifiable surface, microorganisms are less prone to developing resistance toward AuNPs [21]. For example, the kanamycin (Kan)-resistant bacteria (S . bovis , S . epidermidis , E . aerogenes , P . aeruginosa and Y . pestis ) showed increased susceptibility toward Kan-reduced AuNPs. The MIC values for Kan-AuNPs on the test bacteria were significantly reduced to < 10 µg/mL when compared to the MIC values for Kan alone at 50–512 µg/mL. This shows that AuNPs can restore the potency of antibiotics toward the drug-resistant strains by facilitating the uptake and delivery of the antimicrobial agents [21]. AuNPs can enhance drug-loading capacity and control the rate at which the drugs are released. AuNP hybrids with the multi-block copolymers increased the loading capacity of rifampicin and the drug’s half-life to 240 h. By sustaining the drug in the system for that long, ensured slow release of rifampicin from AuNPs at the target sites after oral administration of the AuNP conjugates to rats for 15 days. The drug on the surface was released within 24 h followed by the drug trapped in the polymer matrix after 100 h. And lastly, the drug entrapped between the AuNPs and the polymer matrix took over 240 h to be released in the interstitial space [153].

The AuNP hybrids also allow for the conjugation of multiple molecules with independent but synergistic functions. This was demonstrated by co-functionalization of the AuNPs with antimicrobial peptide (LL37) and the pcDNA that encode for pro-angiogenic factor (vascular endothelial growth factor, VEGF) and used in the treatment of MRSA-infected diabetic wounds in mice [132]. The AuNPs served dual functions, as a vehicle for the biomolecules, and also as transfection agent for the pcDNA. After topical application of the AuNP conjugates on the wound, the LL37 reduced MRSA colonies, while the pcDNA promoted wound healing by inducing angiogenesis through the expression of VEGF [132].

AuNPs have been shown to confer activity and repurpose some non-antibiotic drugs toward antimicrobial activity. The examples of repurposed drugs, which were used for the treatment of diseases other than bacterial infections, include 5FU [58], metformin [147] and 4,6-diamino-2-pyrimidinethiol (DAPT) [13, 112]. AuNPs as drug carriers are able to transport the drugs into the cells and allow direct contact with cellular organelles that resulted in their death [58, 147]. 5FU is an anti-leukemic drug, when attached to AuNPs was shown to kill some bacterial (Micrococcus luteus , S. aureus , P. aeruginosa , E. coli ) and fungal (Aspergillus fumigatus , Aspergillus niger ) strains [58]. While bacteria are resistant to DAPT, DAPT-AuNPs displayed differential antibacterial activity against the Gram-negative bacteria. Furthermore, conjugation of non-antibiotic drugs (e.g., guanidine, metformin, 1-(3-chlorophenyl)biguanide, chloroquine diphosphate, acetylcholine chloride, and melamine) as co-ligands with DAPT on AuNPs exerted non-selective antibacterial activity and a two–fourfold increased activity against Gram-negative bacteria [13]. When used in vivo, orally ingested DAPT-AuNPs showed better protection by increasing the intestinal microflora in E. coli -infected mice. After 4 weeks of treatment, the DAPT-AuNPs cleared the E. coli infection with no sign of mitochondrial damage, inflammation (increase in firmicutes ) or metabolic disorders (reduction in bacteroidetes ) in the mice [112].

The virucidal effects of the AuNP-based systems have been reported against several infectious diseases caused by influenza, measles [154], dengue [155, 156] and human immunodeficiency [115] viruses. Their anti-viral activity was attributed to the ability of AuNPs to either deliver anti-viral agents, or the ability to transform inactive molecules into virucidal agents [154, 156]. AuNPs synthesized using garlic water extracts inhibited measles viral growth in Vero cells infected with the measles virus. When the cells were exposed to both the virus and AuNPs at the same time, they blocked infection of Vero cells by the measles virus [154]. The AuNPs were nontoxic to the Vero cells up to a concentration of 100 µg/mL but inhibited viral uptake by 50% within 15–30 min at a concentration of 8.8 μg/mL [154]. Based on the Plaque Formation Unit assay, the viral load was reduced by 92% after 6 h exposure to 8.8 μg/mL of the AuNPs. The AuNPs interacted with the virus directly and blocked its transmission into the cells [154]. Modification of the AuNP surface with ligands that bind to the virus [156] or anti-viral agents [115, 155] protected them from degradation, enhanced their uptake and delivery onto the cells. The charge of the AuNPs also played a role, with cationic AuNPs being more effective in the delivery and efficacy of the AuNPs than the anionic and neutrally charged NPs. Cationic AuNPs complexed with siRNA inhibited dengue virus-2 replication in dengue virus-2-infected Vero and HepG-2 cells and also the virus infection following pre-treatment of the virus with AuNPs [155]. Inactive molecules are transformed into highly potent anti-viral agents after conjugation to AuNPs. One such example is the transformation of SDC-1721 peptide, a derivative of TAK-779, which is an antagonist of CCR5 and CXCR3 receptors for HIV-1 strain. SDC-1721 has no activity against the HIV-1, but when conjugated to the AuNPs it inhibited HIV-1 infection of the human phytohemagglutinin-stimulated peripheral blood mononuclear cells. The inhibitory effects of SDC-1721-AuNPs were comparable to the TAK-779 [115].

AuNPs as PT Agents

Diseased cells are sensitive to temperatures above 40 °C; cancer cells in particular appear to be even more sensitive to these high temperatures. Studies have shown that high fevers in cancer patients either reduced the symptoms of cancer or completely eradicated the tumors as a result of erysipelas infections [33, 157, 158]. Historically, fevers induced by bacterial infections, hot desert sand bath, or hot baths were used to increase the body temperature in order to kill the cancer cells [157]. These findings gave birth to PT therapy (PTT), which is mostly used for the treatment of cancer. PTT makes use of organic photosensitizers (indocyanine green, phthalocyanine, heptamethine cyanine) that are irradiated by the external source to generate heat energy that will increase the temperature to 40–45 °C (hyperthermia) in the target cells. Hyperthermia then triggers a chain of events (such as cell lysis, denaturation of the genetic materials and proteins), resulting in the destruction of the diseased cells [57, 158,159,160].

The organic dyes are used alone, or in combination with chemotherapy and radiotherapy for enhanced efficacy [157, 160]. Ideally, the effects of the PT agents must be confined to target cells and display minimal bystander effects. However, the organic PT dyes have several limitations such as toxic bystander effects, susceptibility to photobleaching and biodegradation [159]. In recent years, AuNPs are being explored as alternative PT agents as they exhibit strong plasmonic PT properties, and depending on their shape, they can absorb visible or NIR light. Absorption of light in the NIR spectrum is an added advantage that can allow deep tissue PTT [158, 161, 162]. Unlike organic dyes, AuNPs operate in an optical window where the absorption of light by interfering biological PT agents such as hemoglobin, melanin, cytochromes and water is very low [158, 161, 162].

The practicality of AuNP-based PTT has been demonstrated through in vitro and in vivo studies [158, 162, 163]. When the AuNPs are exposed to light, they can convert the absorbed light energy into thermal energy within picoseconds [57, 158, 159], consequently activating cell death via necrosis or apoptosis in the target cells or tissues. AuNP-based hyperthermia in diseased cells has been reported to occur at half the amount of the energy required to kill normal cells, thus perceived to be safer and better PT agents than the conventional dyes [33, 160]. AuNPs can be easily modified to have localized and enhanced PT activity by targeting and accumulating in only diseased cells through either active or passive targeting. And since the tumor environment is already hypoxic, acidic, nutrient starved and have leaky vasculature, the tumors will be most sensitive to the AuNP-based hyperthermia than the surrounding healthy cells and tissues [33, 160].

AuNP-based PTT has been extensively studied [158, 161, 162] and established that AuNPs (e.g., AuNRs, nanocages and nanoshells) that absorb light in the NIR spectrum are best for in vivo and deep tissue PTT [161]. While the ones that absorb and emit light in the visible spectrum (AuNSs and hollow AuNPs) have been demonstrated to treat diseases that affect shallow tissues (up to a depth of 1 mm), which could be of benefit to superficial tumors [158, 161, 162], ocular surgery [164, 165], focal therapy and vocal cord surgery [158, 165]. Although the PTT effects of AuNSs are limited in vivo or for use in deep tissues, combination therapy or active targeting can be incorporated to facilitate target-specific effects [158, 161, 163]. The AuNPs in the combination therapy will serve dual functions as both drug sensitizer and a PT agent, and was shown to enhance anticancer effects of chemotherapeutic drugs [158, 162, 163]. AuNS-Dox combination demonstrated enhanced cancer cell death after laser exposure when compared to the individual effects of the AuNSs and Dox with and without laser treatment [158].

Active targeting on its own can also improve AuNP uptake, localization and target-specific PT effects, which can be viewed in real time by adding fluorophores. AuNSs (25 nm) loaded with transferrin targeting molecules and FITC were shown to accumulate and destroy human breast cancer cells at a higher rate than in non-cancer cells and had better efficacy than the untargeted AuNSs [57]. An independent study also demonstrated that DNA aptamers (As42)-loaded AuNSs (As42-AuNP) induced selective necrosis in Ehrlich carcinoma cells that express HSPA8 protein, a receptor for the aptamers. None of these effects were observed in blood and liver cells mixed with target cells, or cells treated with the AuNSs without laser treatment [163]. The PT effects of the As42-AuNP were replicated in mice transplanted with Ehrlich carcinoma cells in their right leg. As shown in Fig. 9, tail-vein injections of As42-AuNPs followed by laser irradiation resulted in targeted PT destruction of the cancer cells. The As42-AuNPs reduced tumor size in a time-dependent manner; cell death was attributed to increased temperature up to 46 °C at the tumor site. The tumor in mice treated with As42-AuNPs without laser treatment and the AuNPs conjugated with nonspecific DNA oligonucleotide continued to grow but at the lower rate compared to mice injected with PBS. This suggests that the AuNPs were also localized in the tumor [163]. In cases where AuNSs are not efficient for deep tissue PTT, other shapes such as nanocages, nanoshells and AuNRs can be used [158]. Alternately, the visible light absorption of the AuNSs can be shifted to NIR by using processes such as two-photon excitation [57].

In vivo plasmonic PT therapy of cancer cells using targeted AuNSs. As42-AuNPs localized in HSPA8-expressing tumor cells after i.v injection. Exposure to laser treatment resulted in hyperthermia that caused cancer cell death. Reproduced with permission [163]. Copyright 2017, Elsevier

The PT effects of the AuNPs have also been reported for the reversal of obesity [52, 56], using hollow AuNSs (HAuNSs) [52] and AuNRs [56] for the PT lipolysis of the subcutaneous white adipose tissue (sWAT) in obese animals. The HAuNSs were modified with hyaluronate and adipocyte targeting peptide (ATP) to produce HA–HAuNS–ATP conjugate [52]. Hyaluronate was used to ensure topical entry of the HA–HAuNS–ATP through the skin [52, 166], while ATP will recognize and bind to prohibitin once the HAuNSs are internalized. Prohibitin is a receptor that is differentially expressed by the endothelial cells found in the WAT vasculature of obese subjects [5, 52, 55]. The HA–HAuNS–ATP was topically applied in the abdominal region of the obese mice, and through hyaluronate were transdermally shuttled through the epidermis into the dermis where the ATP located the sWATs (Fig. 10) . Illumination of the target site with the NIR laser selectively induced PT lipolysis of the sWAT in the obese mice and reduced their body weight [52]. The AuNRs were used in the photothermolysis-assisted liposuction of the sWATs in Yucatan mini pigs. The untargeted PEG-coated AuNRs (termed NanoLipo) were injected in the sWATs through an incision, followed by laser illumination to heat up the sWATs, which was then aspirated using liposuction. The amount of fat removed from NanoLipo-treated porcine was more than the one removed with conventional suction-assisted lipectomy (SAL). NanoLipo-assisted fat removal had several advantages over the conventional SAL; it took less time (4 min) for liposuction compared to 10 min for SAL, the swelling in the treated site healed faster, and the weight loss effects lasted over 3 months post-liposuction [56].

PT lipolysis of the sWATs using HA-HAuNS-ATP. The ATP was conjugated to the AuNSs for targeted delivery and destruction of the prohibitin-expressing sWATs after NIR laser exposure. Reproduced with permission [52]. Copyright 2017, American Chemical Society

AuNP-based PTT clearly offers a lot of advantages compared to the conventional agents. Their biocompatibility allows for broader applications both in vitro and in vivo. Moreover, they can be customized based on their shapes for shallow (AuNSs) [158, 161, 162] or deep tissue (AuNRs and stars) PTT [158, 161]. At 1–100 nm diameter, AuNPs and its conjugates can circulate long enough to reach and accumulate in the target tissues, with or without targeting moieties [159, 167]. Active targeting can be used to ensure localized PT effects through various routes of administration and might be effective for solid and systemic diseases. AuNP-based PTT can also be used to sensitize cancer cells when administered in combination with chemotherapy, gene therapy and immunotherapy [159]. Therefore, AuNP-based PTT has potential for treatment of chronic diseases [161].