진단 및 치료 응용 프로그램 개선을 위한 다기능 금 나노입자:검토

AuNP의 생물학적 적용

의학에서 AuNPs의 역할과 중요성은 의심할 여지 없이 더 가시화되고 있으며, 이는 광범위한 생물의학 분야에서 AuNP의 다면적 적용을 입증하는 연구의 증가로 뒷받침됩니다. AuNPs의 생체 적합성은 기원전 2500-2600년으로 거슬러 올라가는 인간 질병 치료에서 금의 오랜 역사에 기인합니다. 중국인과 인도인은 남성 발기 부전, 간질, 매독, 류마티스 질환 및 결핵 치료에 금을 사용했습니다. 중국은 회춘과 활력을 위한 아유르베다 의학의 일부로 인도에서 여전히 시행되고 있는 적색 콜로이드 금의 장수 효과를 발견했습니다. Cinnabar-gold(Makaradhwaja라고도 함)는 인도에서 다산 개선에 사용됩니다. 서구 국가에서는 금은 신경 장애와 간질을 치료하는 데 사용되었습니다. 시험관 내 및 생체 내 연구 모두에서 사용에 대한 독성이 보고되지 않았습니다[8, 44, 45]. 그 이후로 경구 및 주사 가능한 금 화합물은 관절염 치료제로 계속 사용되었으며[9, 46], 항암 효과도 있는 것으로 나타났습니다[8]. 질병 진단[47,48,49] 및 치료[3, 29, 50, 51]를 위한 유망한 약제로 부상하고 있는 AuNPs에 대해서도 유사하고 일부 경우에 개선된 효과가 보고되었습니다.

AuNPs는 원하는 기능에 맞게 다양한 생체 분자를 부착하여 생물 의학 응용 분야에 활용할 수 있는 더 큰 표면적을 가지고 있습니다. 여기에는 질병 특이적 바이오마커를 인식하는 데 도움이 되는 표적 부분, 바이오 이미징을 위한 조영제 및 질병 치료를 위한 치료제가 포함될 수 있습니다[24, 25]. 다른 나노 물질에 비해 AuNP를 사용하는 이점은 그림 2[4, 26]에서 보여지는 것처럼 다양한 화학 물질을 사용하여 쉽게 기능화할 수 있다는 것입니다. AuNP는 thiolated 분자에 대해 높은 친화력을 가지며, thiol-gold 결합은 NP 표면에 분자를 흡착하는 데 가장 일반적으로 사용되는 방법입니다[4]. 비오틴-스트렙타비딘 결합 및 카르보디이미드 커플링과 같은 친화도 기반 화학도 사용됩니다. AuNP는 의약의 세 가지 주요 영역인 의약품 전달, 진단 및 치료 목적[24, 35]에서 사용되며 아래에서 설명하는 것처럼 이러한 영역에서 엄청난 잠재력을 보여주었습니다.

AuNP의 합성 및 기능화. 작용기가 있는 생체 분자는 먼저 금-티올 친화력을 통해 NP 표면에 흡착됩니다. 그런 다음, 아민 그룹과 같은 다른 작용기를 사용하여 분자를 카르복실 그룹과 결합하여 표적화 또는 약물 모이어티를 부착할 수 있습니다. [32]에서 수정

약물 전달 에이전트로서의 AuNPs

AuNPs의 가장 일반적인 적용은 약물[11, 18, 52], 백신[53] 및 유전자 요법[24, 32]의 전달 수단입니다. AuNP는 약물 내성, 낮은 약물 분포, 생분해 및 조기 약물 제거와 같은 기존 치료법과 관련된 대부분의 문제를 해결할 수 있는 특성을 가지고 있습니다[11]. AuNPs는 약물 투여량, 치료 빈도를 현저히 감소시킬 수 있으며 소수성 및 불용성 약물을 운반할 수 있습니다. 그들은 생물학적 불활성으로 간주되며 면역 세포의 공격으로부터 화물을 가릴 수 있고 순환계를 통과할 때 약물이 단백질 분해 분해로부터 보호하여 약물 순환 시간을 늘릴 수 있습니다. 이러한 요인은 정상 조직에 거의 또는 전혀 영향을 미치지 않으면서 병든 조직에 약물을 농축 및 유지함으로써 약물의 효능을 쉽게 증가시킬 수 있습니다[25].

암 치료에서 AuNPs의 사용은 광범위하게 연구되어 왔으며[17, 37, 54], 수년에 걸쳐 비만[50, 55, 56] 및 여드름[18]과 같은 다른 질병으로 확장되었습니다. 나노 기반 시스템은 대부분의 세포 구성 요소보다 작으며 병든 조직의 혈관 구조에 대한 EPR(Enhanced Permeability and Retention) 효과를 이용하여 세포 장벽을 수동적으로 횡단할 수 있습니다[25]. 병리학적 상태의 EPR은 과도한 혈관신생 및 투과성 매개체의 증가된 분비를 특징으로 하며, 이는 병든 조직에 의한 AuNP 흡수를 향상시킬 수 있습니다. 이러한 특성은 AuNP 접합체의 선택적 표적화 기회를 제공하는 정상 조직이 아닌 병리학적 상태에만 연관됩니다[25]. AuNP는 여러 분자를 동시에 운반하여 특성을 더욱 다양화할 수 있으므로 약물 운반체로서 매력적입니다. 이것은 AuNP가 특정 생체의학적 기능에 맞게 조정될 수 있기 때문에 대부분의 AuNP 생체 응용 프로그램이 기반으로 하는 의학에서 바람직한 특성입니다. 이것은 세포 소기관과 상호 작용하는 방식을 제어하는 ​​데 도움이 될 수 있으므로 다양한 질병에 대한 효과적인 진단 및 치료 양식의 향후 개발에 대한 약속을 지킬 수 있습니다[4].

AuNP 기반 진단 시스템

나노기술의 출현은 기존의 진단 테스트에 비해 빠르고 강력하며 민감하고 경쟁이 치열한 탐지 시스템 개발에 대한 입장을 높였습니다[48]. 나노물질은 일반적으로 질병의 발병과 관련된 가스, DNA 및 단백질 마커의 검출을 위해 기존 바이오센싱 플랫폼에 통합됩니다[47]. 진단에 사용되는 다양한 나노물질(금속, 고분자, 자기 및 반도체 NP 포함) 중에서 AuNP는 바이오센서, 전기화학 센서 및 발색 분석에 널리 사용되어 질병 바이오마커의 존재를 감지하거나 감지합니다[49]. 국소 SPR(LSPR), 형광 공명 에너지 전달(FRET), 표면 강화 라만 산란, 전도도, 산화 환원 활성 및 양자화된 전하 효과로 인해 표적 분자의 이미징 및 검출에 이상적인 도구입니다[10, 24]. 전자 및 광학 특성, 가시광선 및 근적외선(NIR) 빛을 산란시키는 능력은 현미경 기술(전자, 공초점 및 암시야 광산란)[57], 컴퓨터 단층촬영(CT)과 같은 다양한 기술과 호환되고 측정 가능합니다. , PT 헤테로다인 이미징 기술, UV-Vis 및 라만 분광법 [24, 35].

AuNP 기반 진단 시스템의 개발은 예를 들어 질병 바이오마커를 인식하는 생체 분자의 부착을 통한 AuNP 표면의 변형을 포함합니다[3, 24, 58]. 측면 유동 분석(LFA)은 아마도 나노기술 기반 진단 도구의 가장 잘 알려진 예일 것입니다. LFA는 일반적으로 약 30-40nm의 AuNP를 사용하는데, 그 이유는 더 작은 입자는 소광 단면적이 매우 작은 반면, 더 큰 입자는 일반적으로 이러한 분석에 사용하기에 불안정하기 때문입니다[59]. 또한 AuNP의 SPR, 전도도 및 산화환원의 변화를 유발할 수 있는 다른 분자/효소가 포함됩니다. 이러한 지표는 AuNP 접합체에 분석물이 결합한 후 감지 가능한 신호를 제공하며[24], 신호의 부족 또는 존재는 표적 분자 또는 질병의 부재 또는 존재를 반영합니다. AuNPs에 의해 생성된 신호는 시험관, 스트립, 시험관 내 및 생체 내와 같은 다양한 시험 형식에서 사용될 때 화학적으로 안정적이고 오래 지속되며 일관됩니다[24]. 따라서, 그들의 적용은 진단 분석의 속도와 성공을 현저하게 증가시켰습니다.

비색 AuNP 기반 분석

비색 분석에서 AuNP는 고급 기기를 사용하지 않고도 육안으로 감지할 수 있는 시각적 신호(일반적으로 색상 변화)를 생성합니다. 일반적으로 AuNPs의 콜로이드 용액은 입자간 거리에 크게 의존하는 루비 레드에서 포도색을 띠고 있습니다[60, 61]. 분자 생체 인식 요소(예:항체, 펩타이드, 앱타머, 효소 등)로 변형된 AuNP에 대한 분석물의 결합은 LSPR의 뚜렷한 이동을 유도하여 결과적으로 색상이 루비 레드에서 블루로 변경됩니다[60 , 62, 63]. 색상의 강도는 분석물의 농도에 정비례하며 질병의 존재와 상태를 확인하는 데 사용됩니다. AuNP 기반 비색 진단은 인플루엔자 A 바이러스[64], 지카 바이러스[65], T7 박테리오파지[66], 결핵균의 검출에 성공적으로 사용되었습니다. [67], 그리고 최근에는 중증급성호흡기증후군-코로나바이러스-2(SARS-CoV-2)[60, 68]의 검출을 위해.

비색 AuNP 기반 분석의 예는 전염성이 높은 코로나 바이러스 질병 2019(COVID-19)를 유발하는 바이러스인 SARS-CoV-2[60]의 검출에 대해 입증되었습니다[60, 68]. 이 분석에서 바이러스의 존재는 단순한 색 변화로 보고되었습니다. 진단을 수행하기 위해 어떤 도구도 필요하지 않았습니다. 이 바이러스의 현재 임상 진단 테스트는 4-6시간이 소요되는 역전사효소 실시간 중합효소 연쇄 반응(RT-PCR) 분석을 사용하는 반면, 신속한 현장 진료(PoC) 시스템은 시간이 걸릴 수 있는 항체를 감지합니다. 혈액에 나타나는 며칠. 이에 비해 비색 AuNP 기반 분석은 그림 3에 나와 있는 것처럼 더 강력하고 빠릅니다. SARS-CoV-2 RNA 샘플의 존재 하에 안티센스 올리고뉴클레오티드(ASO)로 태그가 지정된 AuNP를 배양하면 내부에 파란색 침전물이 형성됩니다. ~ 10분 SARS-CoV-2 양성 테스트에서 ASO가 바이러스의 뉴클레오캡시드 인단백질에 있는 N-유전자에 결합하면 시각적으로 감지되는 파란색이 유도되었습니다. 이 검사는 매우 민감했으며 SARS-CoV-2 RNA에 대한 검출 한계가 0.18ng/μL였습니다[60].

AuNP 기반 비색 진단 시스템. ASO로 덮인 AuNP에 의한 SARS-CoV-2 RNA의 선택적 육안 검출. 허가를 받아 재생산 [60]. Copyright 2020, ACS 나노

AuNP 기반 LFA는 그림 3에 표시된 것과 동일한 원리를 따릅니다. 그러나 용액의 색상 변화 대신 분석 물질이 존재할 때 검사 스트립에 가시선이 형성됩니다. 분석 물질이 있는 경우 AuNP가 테스트 라인에 포착되어 육안으로 가시화되는 뚜렷한 빨간색 라인을 형성했습니다. 선의 강도는 흡착된 AuNP의 수에 의해 결정됩니다[69]. Pneumocystis jirovecii 검출에 대한 간단하고 신속한 AuNP 기반 LFA의 예가 그림 4에 나와 있습니다. (P. jirovecii ) 인간 혈청의 IgM 항체. 40nm AuNP는 P의 재조합 합성 항원(RSA)과 접합되었습니다. 지로베치 , P의 존재 또는 부재에 대한 지표로 사용된 주요 표면 당단백질 또는 켁신 유사 세린 프로테아제. 지로베치 . 양성 테스트에서 P. 지로베치 IgM은 접합체 패드에서 AuNP-RSA 접합체에 의해 포착되었습니다. 그런 다음 AuNP-RSA/IgM 복합체는 항-인간 IgM(테스트 라인)에 결합하는 분석 막으로 흘러가고 과잉은 항-RSA 항체(대조군)로 이동하여 두 개의 빨간색 라인이 생성됩니다. 음성 테스트는 대조선에 빨간색만 표시됩니다[70]. 독립적인 연구에서는 AuNP 기반 LFA를 사용하여 SARS-CoV-2 IgM을 선택적으로 검출했으며, 이는 테스트 라인과 컨트롤 라인 모두에서 빨간색 선이 나타나는 것으로 확인되었습니다[68]. 색상은 두 시스템에서 15분 이내에 육안으로 육안으로 감지되었으며 테스트당 10-20μL의 혈청 샘플만 필요했습니다[68, 70].

IgM P 검출을 위한 AuNP 기반 LFA 지로베치 항체. P의 존재(양성 테스트) 또는 부재(음성 대조군). 지로베치 항체는 테스트 라인과 컨트롤 라인 모두에서 AuNP 붉은 색으로 구별되거나 컨트롤 라인에서만 각각 구별될 수 있습니다. 허가를 받아 재생산 [70]. Copyright 2019, 미생물학의 개척지

AuNP를 LFA의 신호 전달 프로브로 사용한 첫 번째 예 중 하나는 Ramos 세포의 검출이었습니다. TE02 앱타머는 캡처 프로브로 사용되었고 TD05 앱타머는 검출 프로브로 사용되었습니다. 앱타머-AuNP 바이오센서는 기기 없이 최소 4000개의 라모스 세포를, 휴대용 스트립 리더로 800개 이상의 라모스 세포를 15분 이내에 시각적으로 감지할 수 있다. 이 샌드위치 검출 바이오센서를 사용하여 이 분석법은 인간 혈액에 스파이크된 Ramos 세포를 성공적으로 검출했으며[71] 순환하는 암세포의 정성 및 정량적 검출을 위한 신속하고 민감하며 저렴한 시스템을 개발하기 위한 개념 증명으로 사용되었습니다. 이후 다양한 AuNP 기반 LFA가 폐포자충증으로 인한 질병을 비롯한 수많은 감염성 질환의 진단을 위해 설계되었습니다. [70], 에볼라 바이러스 [72], HIV, C형 간염 바이러스 및 결핵균 [73] 그리고 더 최근에는 SARS-CoV-2 바이러스[68].

AuNP 기반 이미징 시스템

AuNP는 최대 10 ⁵ 의 공명 파장과 일치하는 빛을 흡수 및 산란하는 능력으로 인해 바이오 이미징 응용 분야에 대해 집중적으로 조사되었습니다. 기존 형광단보다 몇 배나 많습니다[74]. AuNP는 원자 번호와 전자 밀도가 더 높습니다(79 및 19.32g/cm ³ ). ) 기존 요오드 기반 제제(53 및 4.9g/cm ³ ), 따라서 더 나은 조영제임이 입증되었습니다[24]. AuNPs는 병든 세포 또는 조직에 축적되고 강력한 X선 감쇠를 유도하여 표적 부위를 매우 뚜렷하고 쉽게 검출할 수 있게 합니다. AuNP는 특정 항원을 선택적으로 표적화하여 CT 영상화를 위한 뚜렷하고 표적 특이적 대조를 유도할 수 있는 화학적 모이어티 및 분자 생체 인식제에 부착됩니다[75].

시험관 내 표적 분자 CT 이미징 시스템은 전립선 특이적 막 항원(PSMA)에 결합하는 RNA 앱타머로 기능화된 AuNP를 사용하여 달성되었습니다. AuNP-PSMA 앱타머 접합체는 표적 수용체가 없는 PC-3 전립선 세포에 비해 PSMA 발현 전립선(LNCaP) 세포에 대해 4배 이상의 CT 강도를 나타냈다[76]. 유사하게, AuNP-디아트리조산-AS1411 앱타머 접합체는 CL1-5(인간 폐 선암종) 세포 및 CL1-5 종양 보유 마우스에 국한되었다. AS1411 앱타머는 세포 표면의 CL1-5 세포에 의해 발현되는 뉴클레올린(NCL) 수용체를 표적으로 하는 반면, 디아트리조산은 요오드 기반 조영제이다. AuNP-디아트리조산-AS1411 앱타머 접합체는 0.027mM Au Hounsfield 단위(HU ⁻¹ ) 종양 부위에 AuNP가 축적되었음을 나타냅니다[77]. AuNPs는 더 긴 혈관 체류 시간을 나타내어 혈액 내 순환 시간을 연장하고[77,78,79] 디아트리조산의 CT 신호를 개선했습니다[77].

그림 5는 설치류의 심근경색증에서 콜라겐 I을 표적으로 하기 위해 콜라겐 결합 접착 단백질 35(CNA35)에 접합된 AuNP를 사용한 관상 동맥의 생체 내 CT 혈관 영상을 보여줍니다. AuNP 신호는 정맥 주사(i.v) 투여 후 6시간 후에 여전히 혈액에서 검출되었으며, 이는 요오드 기반 약제의 반감기(5-10분)보다 훨씬 높았습니다[79]. 이러한 효과는 만노스 발현(DC 2.4 및 RAW 264.7) 세포에서 수용체 매개 흡수 및 비독성을 나타내는 녹색 합성 만난 캡핑된 AuNP를 사용하여 복제되었습니다. 만난으로 덮인 AuNPs는 마우스의 뒷다리에 주사한 후 생체 내에서 슬와 림프절을 선택적으로 표적화했습니다[38]. AuNP 기반 CT 영상은 관상동맥 및 암에 국한되지 않는 다양한 질병의 진단에 중요한 정보를 제공할 수 있다[76, 77, 79, 80,81]. AuNP를 조영제로 사용하는 것은 광음향, 핵 영상, 초음파 및 자기 공명 영상과 같은 다른 영상 시스템에서 가능성을 보여주었습니다. 이러한 시스템은 다른 곳에서 광범위하게 검토됩니다[82, 83].

AuNP를 CT 조영제로 사용하는 생체 내 CT 영상. Mannan-capped AuNP 및 림프절의 CT 영상(A ) 및 심근 흉터 부담의 CNA35 접합 AuNPs CT 영상화(B ). 허가를 받아 재생산 [79]. Copyright 2018, Elsevier

형광 기반 탐지 시스템의 AuNPs

AuNP는 형광제 또는 형광 소광제로 형광 기반 검출 시스템에서 사용됩니다. 크기 ≤ 5 nm에서 AuNP는 양자점(QD)의 속성을 표시하며 그 자리에서 사용할 수 있습니다. Au₅₅ (PPh₃ )₁₂ Cl₆ 1981년에 도입된 나노클러스터는 양자 크기 거동으로 인해 아마도 가장 집중적으로 연구되었을 것입니다[7]. 이후 Au₂₅와 같은 다양한 양자 크기 AuNP(AuNPsQ) (SR)₁₈ , 금₃₈ (SR)₂₄ 및 Au₁₄₄ (SR)₆₀ [84] 우수한 전자 전도체 및 산화환원 매개체이기 때문에 주로 전기화학적 감지에서 연구되었습니다[85].

AuNPsQ 필름 전극은 전립선 특이 항원(PSA) 검출을 위한 초고감도 전기화학적 면역센서의 제작에 사용되었습니다. 면역센서는 10μL의 희석되지 않은 인간 혈청에서 31.5μA mL/ng의 감도와 0.5pg/mL의 PSA 검출 한계를 보였습니다. 면역 분석은 암의 존재와 관련된 수준보다 낮은 바이오마커 농도에서 다중 부분을 포함하는 이전에 보고된 탄소 나노튜브 숲 면역 센서보다 8배 더 나은 성능을 보였습니다. 이와 같이 정상 상태와 질병 상태 모두에서 테스트 바이오마커를 측정하는 데 사용할 수 있습니다. 면역 센서의 성능은 참조 ELISA 방법과 비슷했습니다[86]. AuNPsQ는 또한 다공성 구조의 CaCO₃에 통합되었습니다. 형광성 CaCO₃를 형성하는 구체 /AuNPsQ 하이브리드는 외상성 뇌 손상 및 폐암에 대한 진단 및 예후 바이오마커인 뉴런 특이적 에놀라제 검출을 위한 것입니다. 센서의 감지 한계는 2.0pg mL ⁻¹ 입니다. [87]. 지금까지 여러 AuNP 기반 형광 검출 시스템이 B형 간염[73, 88], A형 인플루엔자[89], 암[90] 및 심장 손상[91]과 관련된 분석물의 검출을 위해 보고되었습니다.

AuNP는 또한 우수한 FRET 기반 소광제입니다[92]. 독특한 광학 특성(안정된 신호 강도 및 광표백 저항성), 크기 및 수정 가능한 능력으로 인해 형광 감지 플랫폼에서 매력적인 프로브가 되었습니다[93, 94]. 더 큰 AuNP(≥ 10–100 nm)는 직접적인 형광 감지에 적합하지 않은 낮은 양자 수율을 갖습니다. 그러나 상대적으로 높은 여기 에너지 상태에서 형광 염료를 소광시키는 능력은 효과적인 광발광 소광제를 만들었습니다[94]. 원칙적으로 형광 나노프로브는 공여체 형광단(염료 또는 QD)과 수용체 AuNP로 구성되며, 근접하게 되면 선택된 형광단의 형광은 AuNP에 의해 소멸된다[94, 95]. 형광 신호의 부족으로 표시되는 표적이 없는 경우, 핵산 프로브는 혼성화되어 루프 구조를 형성하여 형광단과 반대쪽 말단의 소광제를 근접하게 만듭니다. 분석물이 핵산 프로브에 결합하면 AuNP에서 형광단이 변위되어 형광 신호가 발생합니다[24, 94, 96]. 위에서 언급한 특성을 이용하여 AuNP는 시험관내(금 나노구, AuNS) 및 생체내(금 나노막대, AuNR) 검출을 위한 분자 표지에 통합되었습니다. The two molecular beacons were composed of a matriptase cleavage site as a linker between the AuNPs and the fluorophores. The AuNS–molecular beacon was constructed with the fluorescein isothiocyanate (FITC), and the AuNR–molecular beacon had a NIR fluorescent dye (mercaptopropionic acid, 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].

Toxicity of AuNPs

AuNPs can play an important role in medicine, as demonstrated by the preclinical and clinical studies under review. Their full potential in clinical application as both diagnostic and therapeutic agents can only be realized if they do not pose any health and environmental hazards. While their use in vitro appears to be inconsequential, in vivo application can be hampered by their potential toxicity, which could be detrimental to human health. A major concern with their clinical use is that AuNPs are non-biodegradable and their fate in biological systems has not been fully studied [5, 30]. Although AuNPs are considered to be bio-inert and compatible, their properties (size, shape, charge and composition) raise concerns as they can alter their pharmacokinetics when used in biological environment [27, 34, 118]. The toxicity of AuNPs of varying sizes and shapes has been demonstrated in animals [27, 118]. These NPs can accumulate in the RES organs where they induce damage.

AuNPs are 1–100 nm in diameter which makes them smaller than most of the cellular components. At these sizes, AuNPs can passively transverse cellular barriers and blood vessels by taking advantage of the EPR effect in pathological cells. AuNPs with smaller diameters (1–2 nm) can easily penetrate cell membranes and biologically important cellular organelles such as mitochondria and nuclei [7, 168]. Accumulation of AuNPs in these organelles induces irreversible damage that can cause cellular demise. On the contrary, AuNPs larger than 15 nm are restricted to the cytoplasmic spaces and unable to penetrate internal organelles [168]. These features are desirable for targeting pathological cells, however, AuNPs can also be taken up by healthy cells and alter their physiology [118]. Administration of AuNP-based therapeutics can be done via different routes (i.e., intranasal, oral, transdermal, i.p or i.v) and transported through blood vessels into different tissues and organs [34, 118]. They are able to pass through the blood brain barrier and the placental barrier [34]. Toxicity is size dependent, with certain sizes of AuNPs being well tolerated, while others could be lethal to healthy tissues. Unfunctionalized AuNSs at 8, 17, 12, 37 nm caused physical changes (i.e., change the fur color, loss of bodyweight, camel-like back and crooked spine) within 14 days of treatment (2 doses of 8 mg/kg/week) in rats [118]. Most (> 50%) of the rats died within 21 days (i.e., after 3 doses), and abnormalities in the RES organs (liver, lungs and spleen) were observed. On the contrary, mice treated with 3, 5, 50 and 100 nm AuNPs were not affected by the NPs and no adverse effects or death occurred throughout the duration (50 days) of the study [118]. In diet-induced obese rats that received i.v injections of 14 nm cAuNPs, the NPs were detected in various tissues after 24 h and were mostly confined to the RES organs [55].

The shape, charge and surface chemistry of AuNPs can influence their toxicity. These factors can determine how AuNPs will interact with the biological systems, their cellular uptake and effects on the cells. AuNSs are readily taken up by cells and proven to be less toxic than other shapes such as rods and stars. AuNP surfaces are charged and will influence how they interact and behave within a biological environment [169]. Cationic AuNPs are likely to be more toxic compared to neutral and anionic AuNPs, as their charge allows these NPs to easily interact with negatively charged cell membranes and biomolecules such as DNA. Both the positively and negatively charged AuNPs have been associated with mitochondrial stress, which was not observed with the neutrally charged AuNPs [34, 35].

The shell that forms on the surface of the AuNP core can also influence the functioning of the NPs. These are usually reducing and/ or stabilizing agents such as citrate and CTAB, and once subjected to a biological environment, these molecules can cause either the desorption or absorption of biomolecules found in the biological environment. This can result in the formation of a corona or cause the NPs to become unstable. Citrate- and CTAB-capped AuNPs are highly reactive, which can facilitate the attachment of biocompatible polymers such as PEG, polyvinyl-pyrrolidone, poly (acrylic acid), poly(allylamine hydrochloride), and polyvinyl-alcohol) or biomolecules such as albumin and glutathione to prevent the formation of AuNP-corona with serum proteins. These molecules serve as a stabilizing agent and form a protective layer that can mask the AuNPs from attacks by phagocytes [7, 29, 34, 170] and prevent off-target toxicity [7]. As discussed in “AuNP-Based Therapies” section, AuNPs can be functionalized with targeting and therapeutic agents to define their targets and effects [34].

In addition to their physicochemical properties, the dosage, exposure time and environmental settings also influence the activity of AuNPs. Lower doses and short-term exposure times might render AuNP as nontoxic, while increasing these parameters will lead to cytotoxic effects [34]. Moreover, in vitro studies do not always simulate in vivo studies. At times, AuNPs that seem to be nontoxic in cell culture-based experiments end up being toxic in animal experiments. Many factors could be responsible for these discrepancies [118], and some steps have been identified that can guarantee the safety of AuNPs in biomedical applications. The biocompatibility and target specificity of AuNPs can be improved by modifying the surface of the NPs. Attaching targeting moieties on the AuNPs can channel and restrict their effects to specific targets or pathological cells [5, 55, 127]. Modification of AuNP surface with bio-active peptides provides a platform for developing multifunctional AuNPs with enhanced specificity, efficacy and potentially sustainable effects [11, 127]. All of these effects will be instrumental in the design and development of AuNP-based systems for clinical applications.

Clinical Application of AuNPs

Nanotechnology has the potential to shape the future of healthcare systems and their outcomes. Its promise of creating highly sensitive and effective nanosystems for medicine has been realized with the introduction of organic nanoformulations for cancer treatment. These systems have already paved the way for nanomaterials into clinical applications:doxil and abraxane have been in the market for over two decades and demonstrated the potential of nanotechnology in medicine [1, 2]. More recently, this technology has been used for the development of the SARS-CoV-2 lipid NP-based vaccine to fight against the COVID-19 pandemic [171]. Inorganic nanosystems such as AuNPs offer many advantages over their organic counterparts, yet few of these systems are used clinically (Table 2) [19, 32].

While several AuNP-based drugs are some of the inorganic nanomaterial-based drugs that were tested in clinical trials, they are not progressing at the same rate as organic liposome-based nanodrugs. Aurimune (CYT-6091) and aurolase were the first of AuNP-based formulations to undergo human clinical trials for the treatment of solid tumors. CYT-6091 clinical trials started in 2005 for delivery of recombinant TNF-α as an anticancer therapy in late-stage pancreatic, breast, colon, melanoma, sarcoma and lung cancer patients. CYT-6091 consists of 27-nm cAuNPs loaded with TNF-α and thiolated PEG. The CYT-6091 nanodrug has achieved safety and targeted biologic response at the tumor site at a dose lower than that required for TNF-α alone [16, 17]. CYT-6091 is approved and yet to start phase II clinical trials in combination with chemotherapy. Based on phase II clinical trial strategy, several variants of CYT-6091 have been developed and tested in preclinical studies. All the nanosystems contain TNF-α with either chemotherapy (paclitaxel, dox and gemcitabine), immunotherapy (Interferon gamma) or apoptosis inducing agents attached to the 27 nm cAuNPs [14,15,16]. The AuNP conjugates preferentially accumulated in the tumor sites after systemic administration through the EPR effect and vascular targeting effects of the TNF-α. The AuNPs were not detected in the healthy tissues, and the anti-tumor effects of TNF-α were restricted to the tumor environment [14, 16, 19].

The first clinical trial for the PT treatment with AuroLase® for refractory and/or recurrent head and neck cancers was completed. Information on the outcome of this trial is still pending. The second trial is set to evaluate the effects of AuroLase® on primary and/or metastatic lung tumors in patients where the airway is obstructed [19]. The number of human trials based on AuNP-based formulation is increasing, covering the treatment of a wide range of medical conditions including skin, oral, heart and neurological diseases. AuNP-formulation (150 nm silica-gold nanoshells coated with PEG), which is similar to AuroLase®, was approved for PT treatment of moderate-to-severe inflammatory acne vulgaris. The nanoshells were topically applied on the acne area and transdermally delivered into the follicles and sebaceous ducts through low-frequency ultrasound or massage. Nanoshells applied through massage were effective in penetrating the shallow skin infundibulum (90%) and the sebaceous gland (20%), while the low-frequency ultrasound can penetrate both shallow and deep skin tissues. NIR laser treatment resulted in focal thermolysis of the sebaceous glands in the affected area and disappearance of the acne [18, 167]. The gold–silica nanoshells were well-tolerated, showed no systemic toxic effects with minor side effects (reddiness and swelling) at the treatment site [18]. AuNPs offer many health benefits based on their unique properties but at the same time have raised a lot of political and ethical issues, and resulted in termination of some clinical studies (NCT01436123).

Conclusion and Future Perspectives

Applications of AuNPs in biomedicine are endorsed by their unique physicochemical properties and have shown great promise as theranostic agents. The increasing interest in biomedical applications of AuNPs is further encouraged by the biocompatibility and medical history of bulk gold, which suggests that the gold core in AuNPs will essentially display similar or improved properties [3]. But at the same time their small size can infer unique properties that will completely change their pharmacokinetics [144]. The diverse biomedical applications of AuNPs in diagnostics and therapeutics herein discussed demonstrate their potential to serve as adjunct theranostic agents. They can be used as drug delivery, PTT, diagnostic and molecular imaging agents [12, 33, 128]. In time, and with better knowledge of mechanisms of action, more AuNP-based systems will obtain approval for clinical use. However, the excitement of these biomedical applications of AuNPs should unequivocally be balanced with testing and validation of their safety in living systems before any clinical applications.

In conclusion, more work needs to be done to taper the toxicity of AuNPs. This can be achieved by introducing biocompatible molecules on their surface [14, 15, 58, 159], and developing new and better synthesis methods, such as the use of green chemistry to produce biogenic NPs. All these developments may further broaden the applications of AuNPs in nanomedicine. AuNPs are non-biodegradable, and off-target distribution could result in chronic and lethal effects. All these concerns must be addressed before clinical translation; the existing trials will soon provide some clarity on their impact in human health. Should their health benefits outweigh their potential risks as is the case with the existing clinical drugs, it is a matter of time before they are approved for clinical use.

Availability of Data and Materials

All the information in this paper was obtained from the studies that are already published and referenced accordingly.

Abbreviations

5-FU:

5-Fluorouracil

AD:

Alzheimer's disease

ADDLs:

Amyloid-beta-derived diffusible ligands

ASOs:

Antisense oligonucleotides

AuNPs:

Gold nanoparticles

AuNPsQ:

Quantum-sized AuNPs

AuNRs:

Gold nanorods

AuNSs:

Gold nanospheres

BCA:

Bio-barcoding assay

cAuNPs:

Citrate-capped AuNPs

COVID-19:

Corona virus disease 2019

CSF:

Cerebrospinal fluid

CT:

Computed tomography

CTAB:

Cetyltrimethylammonium bromide

DABCYL:

4-((4′-(Dimethyl-amino)-phenyl)-azo)benzoic acid

DAPT:

4,6-Diamino-2-pyrimidinethiol

Dox:

Doxorubicin

EDC:

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

EPR:

Enhanced permeability and retention

FA:

Folate

FDA:

Food and Drug Administration

FITC:

Fluorescein isothiocyanate

FRET:

Fluorescence resonance energy transfer

HAuNSs:

Hollow AuNSs

HF:

High-fat

HU ⁻¹ :

Hounsfield unit

LFAs:

Lateral flow assays

LSPR:

Localized surface plasmon resonance

MDR:

Multidrug resistant

MGF:

Mangiferin

MPA:

Mercaptopropionic acid

MWPLP:

Microwave-induced plasma-in-liquid process

NCL:

Nucleolin

NIR:

Near-infrared

NSMs:

Nanostructured materials

P. jirovecii :

Pneumocystis jirovecii

PEG:

Polyethylene glycol

PSA:

Prostate-specific antigen

PSMA:

Prostate-specific membrane antigen

PT:

Photothermal

PTT:

Photothermal therapy

QDs:

Quantum dots

RES:

Reticuloendothelial system

ROS:

Reactive oxygen species

SARS-CoV-2:

Severe acute respiratory syndrome-coronavirus-2

SCID:

Severe combined immunodeficiency

SPR:

Surface plasmon resonance

sWAT:

Subcutaneous white adipose tissue

TOAB:

Tetrabutylammonium bromide

TRMs:

Tissue-resident macrophages

VEGF:

Vascular endothelial growth factor

WAT:

White adipose tissue