NADH-depletion triggered energy shutting with cyclometalated iridium (III) complex enabled bimodal Luminescence-SERS sensing and photodynamic therapy.

The nicotinamide adenine dinucleotide-reduced (NADH) function as a hydride (H) carrier to maintain cellular homeostasis. Herein, we report a quinoline appended iridium complex (QAIC) as a molecular probe in fluorescence and surface-enhanced Raman spectroscopy (SERS) modalities to evaluate the endogenous NADH status. NADH-triggered activation of QAIC enabled luminescence (turn-ON) and SERS (turn-OFF) switching phenomenon with a detection limit of 25.6 nM and 15 pM for NADH in luminescence and SERS respectively. Transition state modelling using density functional theory calculations proved that a facile migration of H from NADH to QAIC transformed the activated QAIC (N-QAIC) with an energy span of 19.7 kcal/mol. Furthermore, N-QAIC is probed as a photosensitizer to source singlet oxygen by blocking the photo induced electron transfer (PeT) and generate NAD radicals. Therefore, an efficient light triggered cyclometalated iridium-based molecular probe has been divulged to promote bimodal NADH sensing and multiphase photodynamic therapy.

Dynamic self-assembly of mannosylated-calix[4]arene into micelles for the delivery of hydrophobic drugs.

Carbohydrate-lectin interactions and glycol-molecule-driven self-assembly are powerful yet challenging strategies to create supramolecular nanostructures for biomedical applications. Herein, we develop a modular approach of micellization with a small molecular mannosylated-calix[4]arene synthetic core, CA4-Man3, to generate nano-micelles, CA4-Man3-NPs, which can target cancer cell surface receptors and facilitate the delivery of hydrophobic cargo. The oligomeric nature of the calix[4]arene enables the dynamic self-assembly of calix[4]arene (CA4), where an amphiphile, functionalized with mannose units (CA-glycoconjugates) in the upper rim and alkylated lower rim, afforded the CA4-Man3-NPs in a controllable manner. The presence of thiourea units between calixarene and tri-mannose moiety facilitated the formation of a stable core with bidentate hydrogen bonds, which in turn promoted mannose receptor targeted uptake and helped in the intracellular pH-responsive release of antineoplastic doxorubicin (Dox). Physiochemical features including the stability of the nanomicelle could circumvent the undesirable leakage of the cargoes, ensuring maximum therapeutic output with minimum off-targeted toxicity. Most importantly, surface-enhanced Raman scattering (SERS) was utilized for the first time to evaluate the critical micelle concentration during the formation, cellular uptake and intracellular drug release. The present study not only provides an architectural design of a new class of organic small molecular nanomicelles but also unveils a robust self-assembly approach that paves the way for the delivery of a wide range of hydrophobic chemotherapeutic drugs.

Elucidating a Thermoresponsive Multimodal Photo-Chemotherapeutic Nanodelivery Vehicle to Overcome the Barriers of Doxorubicin Therapy.

In an attempt to circumvent the major pitfalls associated with conventional chemotherapy including drug resistance and off-target toxicity, we have adopted a strategy to simultaneously target both mitochondrial DNA (Mt-DNA) and nuclear DNA (n-DNA) with the aid of a targeted theranostic nanodelivery vehicle (TTNDV). Herein, folic acid-anchored p-sulfo-calix[4]arene (SC4)-capped hollow gold nanoparticles (HGNPs) were meticulously loaded with antineoplastic doxorubicin (Dox) and its mitochondrion-targeted analogue, Mt-Dox, in a pretuned ratio (1:100) for sustained thermoresponsive release of cargo. This therapeutic strategy was enabled to eradicate both n-DNA and Mt-DNA leaving no space to develop drug resistance. The SC4-capped HGNPs (HGNPSC4) were experimented for the first time as a photothermal (PTT) agent with 61.6% photothermal conversion efficiency, and they generated tunable localized heat more efficiently than bare HGNPs. Moreover, the cavity of SC4 facilitated the formation of an inclusion complex with folic acid to target the folate receptor expressing cancer cells and imparted enhanced biocompatibility. The as-synthesized TTNDV was demonstrated to be an ideal substrate for surface-enhanced Raman scattering (SERS) to monitor the molecular-level therapeutic progression in cells and a spheroidal model. A significant reduction in the tumor mass with a marked survival benefit was achieved in syngraft murine models through this synergistic photo-chemotherapy. Collectively, this multifunctional nanoplatform offers a robust approach to treat cancer without any scope of generating Dox resistance and off-target toxicity.

Targeted Theranostic Nano Vehicle Endorsed with Self-Destruction and Immunostimulatory Features to Circumvent Drug Resistance and Wipe-Out Tumor Reinitiating Cancer Stem Cells.

The downsides of conventional cancer monotherapies are profound and enormously consequential, as drug-resistant cancer cells and cancer stem cells (CSC) are typically not eliminated. Here, a targeted theranostic nano vehicle (TTNV) is designed using manganese-doped mesoporous silica nanoparticle with an ideal surface area and pore volume for co-loading an optimized ratio of antineoplastic doxorubicin and a drug efflux inhibitor tariquidar. This strategically framed TTNV is chemically conjugated with folic acid and hyaluronic acid as a dual-targeting entity to promote folate receptor (FR) mediated cancer cells and CD44 mediated CSC uptake, respectively. Interestingly, surface-enhanced Raman spectroscopy is exploited to evaluate the molecular changes associated with therapeutic progression. Tumor microenvironment selective biodegradation and immunostimulatory potential of the MSN-Mn core are safeguarded with a chitosan coating which modulates the premature cargo release and accords biocompatibility. The superior antitumor response in FR-positive syngeneic and CSC-rich human xenograft murine models is associated with a tumor-targeted biodistribution, favorable pharmacokinetics, and an appealing bioelimination pattern of the TTNV with no palpable signs of toxicity. This dual drug-loaded nano vehicle offers a feasible approach for efficient cancer therapy by on demand cargo release in order to execute complete wipe-out of tumor reinitiating cancer stem cells.

Diagnostic spectro-cytology revealing differential recognition of cervical cancer lesions by label-free surface enhanced Raman fingerprints and chemometrics.

Herein we have stepped-up on a strategic spectroscopic modality by utilizing label free ultrasensitive surface enhanced Raman scattering (SERS) technique to generate a differential spectral fingerprint for the prediction of normal (NRML), high-grade intraepithelial lesion (HSIL) and cervical squamous cell carcinoma (CSCC) from exfoliated cell samples of cervix. Three different approaches i.e. single-cell, cell-pellet and extracted DNA from oncology clinic as confirmed by Pap test and HPV PCR were employed. Gold nanoparticles as the SERS substrate favored the increment of Raman intensity exhibited signature identity for Amide III/Nucleobases and carotenoid/glycogen respectively for establishing the empirical discrimination. Moreover, all the spectral invention was subjected to chemometrics including Support Vector Machine (SVM) which furnished an average diagnostic accuracy of 94%, 74% and 92% of the three grades. Combined SERS read-out and machine learning technique in field trial promises its potential to reduce the incidence in low resource countries.

Surface charge modulates the internalization vs. penetration of gold nanoparticles: comprehensive scrutiny on monolayer cancer cells, multicellular spheroids and solid tumors by SERS modality.

Precise control over the dynamics of nanoparticles (NPs) in a tumor microenvironment is highly warranted for the development of an efficient nanotheranostic agent. Even though inductively coupled plasma mass spectrometry can provide a quantitative assessment regarding the uptake efficiency of metal NPs, enumeration of deep tissue penetration capacity remains as a challenge. Herein, we have demonstrated an accurate tracking of the uptake efficiency and penetration phenomenon of gold nanoparticles (AuNPs: 40-50 nm) with respect to three different surface charges in monolayer (2D) cells, multicellular spheroids (3D) and in vivo tumors by surface-enhanced Raman spectroscopy (SERS). While positively charged AuNPs showed around two-fold increased internalization in monolayer cells, SERS-tag-based line scanning on multi-layered tumor spheroids illustrated almost nine-fold superior penetration capability with negatively charged AuNPs. Further, the enhanced solid tumor distribution contributed by the negatively charged AuNPs could appreciably escalate its clinical utility in cancer management.

Biogenic Ag Nanoparticles from Neem Extract: Their Structural Evaluation and Antimicrobial Effects against Pseudomonas nitroreducens and Aspergillus unguis (NII 08123).

Silver nanocrystals have been successfully fabricated by the bioreduction route using the ethanolic extract of Azadirachta indica (neem) leaves as the reducing and capping agent without solvent interference. The silver nanocrystals were grown in a single-step method, without the influence of external energy or surfactants, and at room temperature. The nanoparticles were prepared from different ratios of silver ions to reducing agent molecules and were characterized by UV-vis spectroscopy and transmission electron microscopy (TEM). The nanoparticles were roughly spherical and polydispersed with diameters of less than 40 nm, as determined with high-resolution transmission electron microscopy (HRTEM). Fast Fourier transform (FFT) analysis and X-ray diffraction (XRD) analysis elucidated the crystalline nature of the nanoparticles. The presence of participating functional groups was determined with Fourier transform infrared (FTIR) spectroscopy. The synthesized silver nanoparticles were analyzed as a potential surface-enhanced Raman spectroscopy (SERS) substrate by incorporating rhodamine B as the Raman reporter molecule. The bioreduction process was monitored through SERS fingerprint, which was evaluated by the change in vibrational energies of metal-ligand bonds. It was possible to detect the SERS spectral pattern of the probe molecules on the Ag nanoparticles without the use of any aggregating agent. Thus, the formation of probable intra- and interparticle hot spots was attributed to evaporation-induced aggregation. Furthermore, stirring and precursor salt concentration influenced the kinetics involved in the fabrication process. The thermal stability of the lyophilized nanoparticles prepared from 0.1 M AgNO3 was evaluated with thermogravimetric analysis (TGA) and had a residual mass of 60% at 600 °C. X-ray photoelectron spectroscopy (XPS) studies were used to validate the compositional and chemical-state information. The biomass-capped silver nanoparticles provided antimicrobial activity by inhibiting the growth of Pseudomonas nitroreducens, a biofilm-forming bacterium, and the fungus, Aspergillus unguis (NII 08123).

Tracking the Footprints of Paclitaxel Delivery and Mechanistic Action via SERS Trajectory in Glioblastoma Cells.

The design and development of an efficacious tumor-specific drug-delivery system is a challenging task. In this study, we have synthesized target-specific small peptide substrates on an octaguanidine sorbitol scaffold, named small molecular targeted drug-delivery conjugate (SMTDDC). The SMTDDC fabrication, with dual targeting cRGD and Cathepsin B (Cath B)-specific tripeptide (Glu-Lys-Phe), altered the microtubule network of glioblastoma cells by the orchestrated release of the cytotoxic paclitaxel (PTX). Cath B assisted PTX delivery was monitored by high-performance liquid chromatography and Surface-Enhanced Raman Scattering (SERS) modalities. The time-dependent SERS fingerprinting and imaging revealed a fast and accurate PTX release profile and subsequent in vitro cytotoxicity as well as the apoptotic events and microtubule network alteration in U-87 MG glioblastoma cells. Furthermore, SMTDDC displayed adequate stability under physiological conditions and demonstrated biocompatibility toward red blood cells and lymphocytes. This study indicated a new insight on SERS-guided peptidomimetic sorbitol molecular transporter, enabling a greater promise with high potential for the further development of PTX delivery in glioblastoma treatment.

Exploring Mitochondria-Mediated Intrinsic Apoptosis by New Phytochemical Entities: An Explicit Observation of Cytochrome c Dynamics on Lung and Melanoma Cancer Cells.

Hydnocarpin (Hy) is a flavonoid isolated and purified from the seeds of Hydnocarpus wightiana Blume. Herein, we have developed a built-in semi-synthetic modification on Hy by one-pot multi-component reaction and a [3 + 2] cycloaddition strategy to append five membered isoxazole and isoxazolone as new phytochemical entities (NPCEs). Two selected NPCEs viz Hy-ISO-VIII and Hy-ISO-G from the library of 20 newly synthesized derivatives after in vitro screening unveiled promising cytotoxicity and induced caspase-mediated apoptosis against the human lung and melanoma cancer cells which were well supported by virtual screening based on ligand binding affinity and molecular dynamic simulations. As a new insight, we introduced surface-enhanced Raman spectroscopy to identify the chemo-marker molecular fingerprint to confirm the cellular uptake, cytochrome c release, and DNA fragmentation in a label-free manner. The present findings throw up a surfeit of seminal reasons behind the semi-synthetic modification of Hy, stepping forward to cancer chemotherapy.

Biogenic Cluster-Encased Gold Nanorods as a Targeted Three-in-One Theranostic Nanoenvelope for SERS-Guided Photochemotherapy against Metastatic Melanoma.

Effective treatment of malignant melanoma requires an appropriate combination of therapeutic intervention with long-term prognosis as it often survives by monotherapies. Herein, we report a novel melanoma-targeted theranostic nanoenvelope (MTTNe: ISQ@BSA-AuNC@AuNR@DAC@DR5) which has been constructed by assembling a bovine serum albumin (BSA) stabilized gold nanocluster on a gold nanorod (BSA-AuNC@AuNR), a three-in-one theranostic modality, i.e., photothermal therapy (PTT), photodynamic therapy (PDT), and chemotherapy, tethered with a surface-enhanced Raman scattering (SERS) detection technique. The resultant MTTNe was coloaded with the melanoma-specific FDA approved drug dacarbazine (DAC) and a newly synthesized near-infrared (NIR) absorbing squaraine molecule ISQ that served partly as a photosensitizer and multiplex Raman reporter. Finally, a nanoenvelope was anchored with anti-DR5 monoclonal antibodies as a targeting motif for highly expressed melanoma-specific death receptors in malignant cells. Significant phototherapies of MTTNe were initiated upon an 808 nm single laser trigger which showed a synergistic effect of photothermal hyperthermia as well as singlet oxygen (1O2) driven photodynamic effect in the presence of ISQ followed by on-demand thermoresponsive drug release in the intracellular milieu. Moreover, a multiplex SERS spectral pattern of ISQ (1345 cm-1) and DAC (1269 cm-1) has been utilized for monitoring precise drug release kinetics and target-specific recognition on melanoma cells by Raman imaging. Therapeutic performance of the nanoenvelope was evaluated by in vitro cytotoxicity studies in human melanoma cells (A375) and confirmed the apoptotic phenomenon by molecular-level monitoring of intracellular SERS fingerprints. Finally, to address the biocompatibility of MTTNe, in vivo subacute toxicity was conducted on BALB/c mice. Hence, the current studies mark a footstep of a facile strategy for the treatment of melanoma by synergistic multimodal photothermal/photodynamic/chemotherapy.

Enzyme-Driven Switchable Fluorescence-SERS Diagnostic Nanococktail for the Multiplex Detection of Lung Cancer Biomarkers.

Comprehensive profiling of multiple protein targets plays a critical role in deeper understanding of specific disease conditions associated with high heterogeneity and complexity. Herein, we present the design and fabrication of smart programmable nanoarchitectures, which could integrate clinically relevant diagnostic modalities for the multiplexed detection of most prevalent panel of disease biomarkers present in lung cancer. The multiplex nanoprobes were prepared by attaching dual-functional Raman-active fluorogens onto spherical gold nanoparticles through a peptide linker, Phe-Lys-Cys (FKC), which is engineered with a cathepsin B (cathB) enzyme cleavage site. The presence of cathB induces the scission of FKC upon homing into the cancer cells, resulting in the release of the initially latent fluorophores with a concomitant quenching of the surface-enhanced Raman signal intensity, thereby realizing an on-off switching between the fluorescence and Raman modalities. The enzyme-triggered switchable nanoprobes were utilized for the simultaneous detection of pathologically relevant lung cancer targets by tethering with specific antibody units. The multiplex-targeted multicolor coded detection capability of the antitags was successfully developed as a valid protein screening methodology, which can address the unmet challenges in the conventional clinical scenario for the precise and early diagnosis of lung cancer.

Exploring the margins of SERS in practical domain: An emerging diagnostic modality for modern biomedical applications.

Excellent multiplexing capability, molecular specificity, high sensitivity and the potential of resolving complex molecular level biological compositions augmented the diagnostic modality of surface-enhanced Raman scattering (SERS) in biology and medicine. While maintaining all the merits of classical Raman spectroscopy, SERS provides a more sensitive and selective detection and quantification platform. Non-invasive, chemically specific and spatially resolved analysis facilitates the exploration of SERS-based nano probes in diagnostic and theranostic applications with improved clinical outcomes compared to the currently available so called state-of-art technologies. Adequate knowledge on the mechanism and properties of SERS based nano probes are inevitable in utilizing the full potential of this modality for biomedical applications. The safety and efficiency of metal nanoparticles and Raman reporters have to be critically evaluated for the successful translation of SERS in to clinics. In this context, the present review attempts to give a comprehensive overview about the selected medical, biomedical and allied applications of SERS while highlighting recent and relevant outcomes ranging from simple detection platforms to complicated clinical applications.

Calix[4]arene Based Redox Sensitive Molecular Probe for SERS Guided Recognition of Labile Iron Pool in Tumor Cells.

Targeting the intracellular "labile" iron pool is turned as a key modulator for cancer progression since the former is responsible for several pathological processes in tumor cells. Herein, we report a nonfluorescent calix[4]arene based triazole appended molecular probe (PTBC) for redox-specific detection of Fe3+ under physiological condition by UV-vis, FT-IR, 1H NMR, HR-MS spectroscopies, ITC, and the binding strategy between Calix[4]arene and Fe3+ was modeled by DFT calculations. As a new insight PTBC probe showed significant Raman fingerprint through surface enhanced Raman scattering (SERS) modality revealing the ultrasensitive detection of Fe3+ with a LOD of 2 nM. Interestingly, intracellular "iron pool" has been recognized in human lung adenocarcinoma cells (A549) by the PTBC illustrating the distinct Raman mapping. Finally, PTBC imparted cytotoxicity via reactive oxygen species (ROS) generation in cellular milieu signifies its capability as a theranostic molecular probe.

Plasmonically Enhanced Galactoxyloglucan Endowed Gold Nanoparticles Exposed Tumor Targeting Biodistribution Envisaged in a Surface-Enhanced Raman Scattering Platform.

Biopolymer-capped gold nanoparticles (AuNPs) were perceived for tracing biodistribution in a solid tumor mice through surface-enhanced Raman scattering (SERS) fingerprinting. In this strategy, a robust and ecofriendly green chemistry approach was adopted to construct galactoxyloglucan (PST001) endowed AuNPs (PST-GNPs) with cancer-cell-selective toxic nature and excellent biocompatibility. Plasmonically enhanced light-scattering properties facilitated PST-GNPs to be a superior SERS substrate with high Raman signal enhancement. In this context, PST-GNPs were scrutinized for the noninvasive label-free SERS live-cell spectral imaging to evaluate the fingerprint molecular details of cellular processes. Consequently, the inherent SERS feature of PST-GNPs enabled us to investigate the dynamic and complex nature with NP biodistrubution in tumor-bearing mice on a SERS platform that illustrated the tumor targeting nature. Henceforth, the present findings emphasized a futuristic clinically relevant scenario for tracing the in vivo NP dissemination in a label-free fashion for providing vital biochemical details on a molecular level.

Exploration of Biogenic Nano-chemobiotics Fabricated by Silver Nanoparticle and Galactoxyloglucan with an Efficient Biodistribution in Solid Tumor Investigated by SERS Fingerprinting.

An incredible exploration ensued of a dual modality nanocomposite wherein chemotherapy in fusion with antibacterial efficacy is obtained in a biogenic fabrication, which transformed as a novel nano-chemobiotics (NCB) prevailing fundamental molecular level investigation by surface-enhanced Raman scattering (SERS) platform. The nanocomposite is a facile, robust, and ecofriendly constitution between silver nanoparticles (SNPs) and a naturally occurring galactoxyloglucan (PST001) denoted as SNP@PST, which displayed biocompatibility with an upgraded selective cytotoxicity toward cancer cells. The relatively nontoxic nature of the SNP@PST on normal cells and red blood cells was further proved by detailed toxicological profiling on BALB/c mice. As a unique outcome, we observed excellent antibacterial activity, which is complementary to the greater cytotoxicity by the NCB. In diagnostic aspect, SNP@PST was revealed to be a superior SERS substrate with multiscale Raman signal enhancement contributed by homogeneous hot-spot distribution. Finally, the inherent SERS feature enabled us to investigate the biodistribution of the NCB in tumor-challenged mice using Raman fingerprinting and mapping analysis. Hence, the unrevealed SNP@PST orchestrated with the surfactant-free green method resembled a potential theransonstic NCB construct with synergistic anticancer and antibacterial potential in a single platform.

TEMPO-Oxidized Nanocellulose Fiber-Directed Stable Aqueous Suspension of Plasmonic Flower-like Silver Nanoconstructs for Ultra-Trace Detection of Analytes.

The synthesis of shape-tuned silver (Ag) nanostructures with high plasmon characteristics has become of significant importance in in vitro diagnostic applications. Herein, we report a simple aqueous synthetic route using 2,2,6,6-tetramethylpiperidine-1-oxyl-oxidized nanocellulose fibers (T-NCFs) and trisodium citrate (TSC) that results in anisotropically grown flower-like Ag nanoconstructs (AgNFs). A detailed investigation of the concentration and sequence of the addition of reactants in the formation of these anisotropic Ag structures is presented. Our experimental results show that the mechanism underlying the formation of AgNFs is facilitated by the synergistic action of T-NCFs and TSC on the directional growth of Ag nuclei during the primary stage, which later develop into a flower-like structure by the ripening of larger particles consuming smaller Ag particles. As a result the final structure comprises flower-like morphology over which several smaller Ag particles (of size <10 nm) are adhered. The aqueous AgNF colloid exhibits high stability (ζ = -69.4 mV) and long shelf-life at neutral pH (>4 months) by the efficient capping action of T-NCFs. Further, an as-synthesized nanoconstructs shows excellent surface-enhanced Raman scattering activity, which enables ultrasensitive detection of p-aminothiophenol with a concentration down to 10 aM (10-17 M) in a reproducible way. This biosupported synthesis of stable aqueous colloids of AgNF may find potential applications as a biomedical sensing platform for the trace level detection of analyte molecules.

Investigation of apoptotic events at molecular level induced by SERS guided targeted theranostic nanoprobe.

Herein, we have examined distinctive structural and functional variations of cellular components during apoptotic cell death induced by a targeted theranostic nanoprobe, MMP-SQ@GNR@LAH-DOX, which acted as a SERS "on/off" probe in the presence of a MMP protease and executed synergistic photothermal chemotherapy, as reflected by the SERS fingerprinting, corresponding to the phosphodiester backbone of DNA.

New Insight of Tetraphenylethylene-based Raman Signatures for Targeted SERS Nanoprobe Construction Toward Prostate Cancer Cell Detection.

We have designed and synthesized novel tetraphenylethylene (TPE) appended organic fluorogens and unfold their unique Raman fingerprinting reflected by surface-enhanced Raman scattering (SERS) upon adsorption on nanoroughened gold surface as a new insight in addition to their prevalent aggregation-induced emission (AIE) and aggregation-caused quenching (ACQ) phenomena. A series of five TPE analogues has been synthesized consisting of different electron donors such as (1) indoline with propyl (TPE-In), (2) indoline with lipoic acid (TPE-In-L), (3) indoline with Boc-protected propyl amine (TPE-In-Boc), (4) benzothaizole (TPE-B), and (5) quinaldine (TPE-Q). Interestingly, all five TPE analogues produced multiplexing Raman signal pattern, out of which TPE-In-Boc showed a significant increase in signal intensity in the fingerprint region. An efficient SERS nanoprobe has been constructed using gold nanoparticles as SERS substrate, and the TPE-In as the Raman reporter, which conjugated with a specific peptide substrate, Cys-Ser-Lys-Leu-Gln-OH, well-known for the recognition of prostate-specific antigen (PSA). The designated nanoprobe TPE-In-PSA@Au acted as SERS "ON/OFF" probe in peace with the vicinity of PSA protease, which distinctly recognizes PSA expression with a limit of detection of 0.5 ng in SERS platform. Furthermore, TPE-In-PSA@Au nanoprobe was efficiently recognized the overexpressed PSA in human LNCaP cells, which can be visualized through SERS spectral analysis and SERS mapping.

A Dual-Targeting Octaguanidine-Doxorubicin Conjugate Transporter for Inducing Caspase-Mediated Apoptosis on Folate-Expressing Cancer Cells.

An efficient synthetic framework was assembled (G8-FKE-FA-Dox), consisting of a lysosome-targeting octaguanidine molecular transporter with a cathepsin B (cath B)-specific peptide substrate, folic acid, and the potent chemotherapeutic drug doxorubicin (Dox). Because the folate receptor (FR) and cath B are overexpressed in malignant cells, this transporter conjugate successfully executed lysosome-mediated transport of Dox to FR-positive tumor cells, illustrating this framework as an excellent targeted drug delivery system (TDDS). G8-FKE-FA-Dox was shown to exhibit selective toxicity toward FR-overexpressing cancer cells, with an IC50 value superior to that of the USFDA-approved Lipodox(TM) and proportional to that of free Dox via selective induction of apoptosis by the activation of caspases 8, 9, and 3. This TDDS was observed to be nontoxic to red blood cells and lymphocytes at neutral pH. Furthermore the tumor-targeting dissemination pattern of this system was revealed by monitoring the in vivo biodistribution of the carrier (G8-FKE-FA-FL) in normal and FR-overexpressing tumor-bearing mice.