Cytotoxicity of BSA‐Stabilized Gold Nanoclusters: In Vitro and In Vivo Study

Gold nanoclusters (Au NCs) are one of the most promising fluorescent nanomaterials for bioimaging, targeting, and cancer therapy due to their tunable optical properties, yet their biocompatibility still remains unclear. Herein, the cytotoxicity of bovine serum albumin (BSA)‐stabilized Au NCs is studied by using three tumor cell lines and two normal cell lines. The results indicate that Au NCs induce the decline of cell viabilities of different cell lines to varying degrees in a dose‐ and time‐dependent manner, and umbilical vein endothelial cells which had a higher intake of Au NCs than melanoma cells show more toxicity. Addition of free BSA to BSA‐Au NCs solutions can relieve the cytotoxicity, implying that BSA can prevent cell damage. Moreover, Au NCs increase intracellular reactive oxygen species (ROS) production, further causing cell apoptosis. Furthermore, N‐acetylcysteine, a ROS scavenger, partially reverses Au NCs‐induced cell apoptosis and cytotoxicity, indicating that ROS might be one of the primary reasons for the toxicity of BSA‐Au NCs. Surprisingly, Au NCs with concentrations of 5 and 20 nM significantly inhibit tumor growth in the xenograft mice model of human liver cancer, which might provide a new avenue for the design of anti‐cancer drug delivery vehicles.     

A Highly Entangled Polymeric Nanoconstruct Assembled by siRNA and its Reduction‐Triggered siRNA Release for Gene Silencing

A nanoconstruct (NC) is developed from a biocompatible natural polymer and siRNA conjugates to deliver small interfering RNA (siRNA) target‐specifically without cationic condensation reagents. This study reports a novel siRNA‐mediated cross‐linked NC produced by hybridizing two complementary single‐stranded siRNAs that are conjugated to the polymer dextran via a disulfide linkage. The reducible disulfide bond between the siRNA and polymer allow siRNA release from the NC in the reducible cytoplasmic region after the NC enters the cell. In addition, when the NC contains the prostate‐carcinoma‐binding peptide aptamer DUP‐1, it can selectively deliver siRNA into prostate cancer cells of the PC‐3 lines; thus, the newly formulated NC has reduced the cytotoxicity and improved the efficacy of target‐specific siRNA delivery. Moreover, this new concept of NCs using biocompatible siRNA and a neutral polymer may provide insightful knowledge for future directions for designing NCs for stimuli‐responsive and advanced target‐specific siRNA delivery.     

Passively Targeted Curcumin‐Loaded PEGylated PLGA Nanocapsules for Colon Cancer Therapy In Vivo

Clinical applications of curcumin for the treatment of cancer and other chronic diseases have been mainly hindered by its short biological half‐life and poor water solubility. Nanotechnology‐based drug delivery systems have the potential to enhance the efficacy of poorly soluble drugs for systemic delivery. This study proposes the use of poly(lactic‐co‐glycolic acid) (PLGA)‐based polymeric oil‐cored nanocapsules (NCs) for curcumin loading and delivery to colon cancer in mice after systemic injection. Formulations of different oil compositions are prepared and characterized for their curcumin loading, physico‐chemical properties, and shelf‐life stability. The results indicate that castor oil‐cored PLGA‐based NC achieves high drug loading efficiency (≈18% w(drug)/w(polymer)%) compared to previously reported NCs. Curcumin‐loaded NCs internalize more efficiently in CT26 cells than the free drug, and exert therapeutic activity in vitro, leading to apoptosis and blocking the cell cycle. In addition, the formulated NC exhibits an extended blood circulation profile compared to the non‐PEGylated NC, and accumulates in the subcutaneous CT26‐tumors in mice, after systemic administration. The results are confirmed by optical and single photon emission computed tomography/computed tomography (SPECT/CT) imaging. In vivo growth delay studies are performed, and significantly smaller tumor volumes are achieved compared to empty NC injected animals. This study shows the great potential of the formulated NC for treating colon cancer.     

Functional Graphene Oxide as a Nanocarrier for Controlled Loading and Targeted Delivery of Mixed Anticancer Drugs

A simple synthetic route for the preparation of functional nanoscale graphene oxide (NGO), a novel nanocarrier for the loading and targeted delivery of anticancer drugs, is reported. The NGO is functionalized with sulfonic acid groups, which render it stable in physiological solution, followed by covalent binding of folic acid (FA) molecules to the NGO, thus allowing it to specifically target MCF‐7 cells, human breast cancer cells with FA receptors. Furthermore, controlled loading of two anticancer drugs, doxorubicin (DOX) and camptothecin (CPT), onto the FA‐conjugated NGO (FA–NGO) via π–π stacking and hydrophobic interactions is investigated. It is demonstrated that FA–NGO loaded with the two anticancer drugs shows specific targeting to MCF‐7 cells, and remarkably high cytotoxicity compared to NGO loaded with either DOX or CPT only. Considering that the combined use of two or more drugs, a widely adopted clinical practice, often displays much better therapeutic efficacy than that of a single drug, the controlled loading and targeted delivery of mixed anticancer drugs using these graphene‐based nanocarriers may find widespread application in biomedicine.     

Synthesis and enzymatic cleavage of dual-ligand quantum dots

Site directed therapy promises to minimize treatment-limiting systemic effects associated with cytotoxic agents that have no specificity for pathologic tissues. One general strategy is to target cell surface receptors uniquely presented on particular tissues. Highly specific in vivo targeting of an emerging neoplasm through a single molecular recognition mechanism has not generally been successful. Nonspecific binding and specific binding to non-target cells compromise the therapeutic index of small molecule, ubiquitous cancer targeting ligands. In this work, we have designed and fabricated a nanoparticle (NP) construct that could potentially overcome the current limitations of targeted in vivo delivery. Quantum dots (QDs) were functionalized with a poly(ethylene glycol) (PEG) modified to enable specific cleavage by matrix metalloprotease-7 (MMP-7). The QDs were further functionalized with folic acid, a ligand for a cell surface receptor that is overexpressed in many tumors, but also expressed in some normal tissues. The nanomolecular construct is designed so that the PEG initially conceals the folate ligand and construct binding to cells is inhibited. MMP-7 activated peptide cleavage and subsequent unmasking of the folate ligand occurs only near tumor tissue, resulting in a proximity activated (PA) targeting system. QDs functionalized with both the MMP-7 cleavable substrate and folic acid were successfully synthesized and characterized. The proteolytic capability of the dual ligand QD construct was quantitatively assessed by fluorometric analysis and compared to a QD construct functionalized with only the PA ligand. The dual ligand PA nanoparticles studied here exhibit significant susceptibility to cleavage by MMP-7 at physiologically relevant conditions. The capacity to autonomously convert a biopassivated nanostructure to a tissue-specific targeted delivery agent in vivo represents a paradigm change for site-directed therapies.     

Al Alloy Nanocomposite Reinforced with Physically Functionalized Carbon Nanotubes Synthesized via Spark Plasma Sintering

Nanocrystalline Al–11.5Si (in wt%) powders were prepared by high-energy ball milling of microcrystalline Al–Si powders, which were subsequently mixed with pristine and physically functionalized multiwalled carbon nanotubes (MWCNTs) separately and were consolidated by spark plasma sintering. Improvement in MWCNT dispersion was observed as a result of functionalization, which resulted in improved densification of the nanocomposites (NCs). Scanning electron microscopy was performed to understand the agglomeration and dispersion of CNTs. Distribution of MWCNTs, the dislocation activity, and the effect of primary Si particles in NC matrix were studied by carrying out transmission electron microscopy. Nanoindentation was performed to measure the elastic modulus and microhardness of the NCs which showed appreciable improvement for functionalized MWCNT reinforced NC.     

Modeling particle shape-dependent dynamics in nanomedicine

One of the major challenges in nanomedicine is to improve nanoparticle cell selectivity and adhesion efficiency through designing functionalized nanoparticles of controlled sizes, shapes, and material compositions. Recent data on cylindrically shaped filomicelles are beginning to show that non-spherical particles remarkably improved the biological properties over spherical counterpart. Despite these exciting advances, non-spherical particles have not been widely used in nanomedicine applications due to the lack of fundamental understanding of shape effect on targeting efficiency. This paper intends to investigate the shape-dependent adhesion kinetics of non-spherical nanoparticles through computational modeling. The ligand-receptor binding kinetics is coupled with Brownian dynamics to study the dynamic delivery process of nanorods under various vascular flow conditions. The influences of nanoparticle shape, ligand density, and shear rate on adhesion probability are studied. Nanorods are observed to contact and adhere to the wall much easier than their spherical counterparts under the same configuration due to their tumbling motion. The binding probability of a nanorod under a shear rate of 8 s(-1) is found to be three times higher than that of a nanosphere with the same volume. The particle binding probability decreases with increased flow shear rate and channel height. The Brownian motion is found to largely enhance nanoparticle binding. Results from this study contribute to the fundamental understanding and knowledge on how particle shape affects the transport and targeting efficiency of nanocarriers, which will provide mechanistic insights on the design of shape-specific nanomedicine for targeted drug delivery applications.     

HDL‐Mimicking Peptide–Lipid Nanoparticles with Improved Tumor Targeting

Targeted delivery of intracellularly active diagnostics and therapeutics in vivo is a major challenge in cancer nanomedicine. A nanocarrier should possess long circulation time yet be small and stable enough to freely navigate through interstitial space to deliver its cargo to targeted cells. Herein, it is shown that by adding targeting ligands to nanoparticles that mimic high‐density lipoprotein (HDL), tumor‐targeted sub‐30‐nm peptide–lipid nanocarriers are created with controllable size, cargo loading, and shielding properties. The size of the nanocarrier is tunable between 10 and 30 nm, which correlates with a payload of 15–100 molecules of fluorescent dye. Ligand‐directed nanocarriers targeting epidermal growth factor receptor (EGFR) are confirmed both in vitro and in vivo. The nanocarriers show favorable circulation time, tumor accumulation, and biodistribution with or without the targeting ligand. The EGFR targeting ligand is proved to be essential for the EGFR‐mediated tumor cell uptake of the nanocarriers, a prerequisite of intracellular delivery. The results demonstrate that targeted HDL‐mimetic nanocarriers are useful delivery vehicles that could open new avenues for the development of clinically viable targeted nanomedicine.     

Cancer‐Cell‐Specific Induction of Apoptosis Using Mesoporous Silica Nanoparticles as Drug‐Delivery Vectors

Targeted delivery of the chemotherapeutic agent methotrexate (MTX) to cancer cells using poly(ethyleneimine)‐functionalized mesoporous silica particles as drug‐delivery vectors is reported. Due to its high affinity for folate receptors, the expression of which is elevated in cancer cells, MTX serves as both a targeting ligand and a cytotoxic agent. Enhanced cancer‐cell apoptosis (programmed cell death) relative to free MTX is thus observed at particle concentrations where nonspecific MTX‐induced apoptosis is not observed in the nontargeted healthy cell line, while corresponding amounts of free drug affect both cell lines equally. The particles remain compartmentalized in endo‐/lysosomes during the time of observation (up to 72 h), while the drug is released from the particle only upon cell entry, thereby inducing selective apoptosis in the target cells. As MTX is mainly attached to the particle surface, an additional advantage is that the presented carrier design allows for adsorption (loading) of additional drugs into the pore network for therapies based on a combination of drugs.     

Induction of apoptosis in cancer cells at low silver nanoparticle concentrations using chitosan nanocarrier

We report the development of a chitosan nanocarrier (NC)-based delivery of silver nanoparticles (Ag NPs) to mammalian cells for induction of apoptosis at very low concentrations of the NPs. The cytotoxic efficacy of the Ag NP-nanocarrier (Ag-CS NC) system in human colon cancer cells (HT 29) was examined by morphological analyses and biochemical assays. Cell viability assay demonstrated that the concentration of Ag NPs required to reduce the viability of HT 29 cells by 50% was 0.33 μg mL(-1), much less than in previously reported data. The efficient induction of apoptosis by Ag-CS NCs was confirmed by flow cytometry. Additionally, the characteristic nuclear and morphological changes during apoptotic cell death were investigated by fluorescence and scanning electron microscopy (SEM), respectively. The involvement of mitochondrial pathway of cell death in the Ag-CS NCs induced apoptosis was evident from the depolarization of mitochondrial membrane potential (ΔΨ(m)). Real time quantitative RT-PCR analysis demonstrated the up-regulation of caspase 3 expression which was further reflected in the formation of oligo-nucleosomal DNA "ladders" in Ag-CS NCs treated cells, indicating the important role of caspases in the present apoptotic process. The increased production of intracellular ROS due to Ag-CS NCs treatment indicated that the oxidative stress could augment the induction of apoptosis in HT 29 cells in addition to classical caspase signaling pathway. The use of significantly low concentration of Ag NPs impregnated in chitosan nanocarrier is a much superior approach in comparison to the use of free Ag NPs in cancer therapy.     

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes.     

Endosomal Escape of Polymer‐Coated Silica Nanoparticles in Endothelial Cells

Current investigations into hazardous nanoparticles (i.e., nanotoxicology) aim to understand the working mechanisms that drive toxicity. This understanding has been used to predict the biological impact of the nanocarriers as a function of their synthesis, material composition, and physicochemical characteristics. It is particularly critical to characterize the events that immediately follow cell stress resulting from nanoparticle internalization. While reactive oxygen species and activation of autophagy are universally recognized as mechanisms of nanotoxicity, the progression of these phenomena during cell recovery has yet to be comprehensively evaluated. Herein, primary human endothelial cells are exposed to controlled concentrations of polymer‐functionalized silica nanoparticles to induce lysosomal damage and achieve cytosolic delivery. In this model, the recovery of cell functions lost following endosomal escape is primarily represented by changes in cell distribution and the subsequent partitioning of particles into dividing cells. Furthermore, multilamellar bodies are found to accumulate around the particles, demonstrating progressive endosomal escape. This work provides a set of biological parameters that can be used to assess cell stress related to nanoparticle exposure and the subsequent recovery of cell processes as a function of endosomal escape.     

Precision Nanocluster-Based Toroidal and Supertoroidal Frameworks Using Photocycloaddition-Assisted Dynamic Covalent Chemistry

Atomically precise nanoclusters (NCs) have recently emerged as ideal building blocks for constructing self-assembled multifunctional superstructures. The existing structures are based on various non-covalent interactions of the ligands on the NC surface, resulting in inter-NC interactions. Despite recent demonstrations on light-induced reversible self-assembly, long-range reversible self-assembly based on dynamic covalent chemistry on the NC surface has yet to be investigated. Here, it is shown that Au₂₅ NCs containing thiolated umbelliferone (7-hydroxycoumarin) ligands allow [2+2] photocycloaddition reaction-induced self-assembly into colloidal-level toroids. The toroids upon further irradiation undergo inter-toroidal reaction resulting in macroscopic supertoroidal honey-comb frameworks. Systematic investigation using electron microscopy, atomic force microscopy (AFM), and electron tomography (ET) suggest that the NCs initially form spherical aggregates. The spherical structures further undergo fusion resulting in toroid formation. Finally, the toroids fuse into macroscopic honeycomb frameworks. As a proof-of-concept, a cross-photocycloaddition reaction between coumarin-tethered NCs and an anticancer drug (5-fluorouracil) is demonstrated as a model photo-controlled drug release system. The model system allows systematic loading and unloading of the drug during the assembly and disassembly under two different wavelengths. The results suggest that the dynamic covalent chemistry on the NC surface offers a facile route for hierarchical multifunctional frameworks and photocontrolled drug release.     

Feasibility of obtaining in situ nanocapsules through modified self-microemulsifying drug delivery systems. A new manufacturing approach for oral route administration

Nanocapsules (NCs) are submicron-sized core shell systems which present important advantages such as improvement of drug efficacy and bioavailability, prevention of drug degradation, and provision of controlled-release delivery. The available methods for NC production require expensive recovery and purification steps which compromised the morphology of NCs. Industrial applications of NCs have been avoided due to the aforementioned issues. In this study, we developed a new method based on a modified self-microemulsifying drug delivery system (SMEDDS) for in situ NCs production within the gastrointestinal tract. This new methodology does not require purification and recovery steps and can preserve the morphology and the functionality of NCs. The in situ formed NCs of Eudragit^® RL PO were compared with nanospheres (NEs) in order to obtain evidence of their core-shell structure. NCs presented a spherical morphology with a size of 126.2?±?13.1?nm, an ibuprofen encapsulation efficiency of 31.3% and a zeta-potential of 37.4?mV. Additionally, NC density and release profile (zero order) showed physical evidence of the feasibility of NCs in situ creation.     

Somatostatin Receptor‐Mediated Tumor‐Targeting Nanocarriers Based on Octreotide‐PEG Conjugated Nanographene Oxide for Combined Chemo and Photothermal Therapy

Nano‐sized in vivo active targeting drug delivery systems have been developed to a high anti‐tumor efficacy strategy against certain cancer‐cells‐specific. Graphene based nanocarriers with unique physical and chemical properties have shown significant potentials in this aspect. Here, octreotide (OCT), an efficient biotarget molecule, is conjugated to PEGylated nanographene oxide (NGO) drug carriers for the first time. The obtained NGO‐PEG‐OCT complex shows low toxicity and excellent stability in vivo and is able to achieve somatostatin receptor‐mediated tumor‐specific targeting delivery. Owing to the high loading efficiency and accurate targeting delivery of anti‐cancer drug doxorubicin (DOX), our DOX loaded NGO‐PEG‐OCT complex offers a remarkably improved cancer‐cell‐specific cellular uptake, chemo‐cytotoxicity, and decreased systemic toxicity compared to free DOX or NGO‐PEG. More importantly, due to its strong near‐infrared absorption, the NGO‐PEG‐OCT complex further enhances efficient photothermal ablation of tumors, delivering combined chemo and photothermal therapeutic effect against cancer cells.     

Targeting Nanocarriers with Anisamide: Fact or Artifact?

Encapsulating chemotherapeutics in nanoparticles can reduce the side effects of intravenous administration and improve their antitumor efficacy. Additionally, surface decoration of the nanocarriers with tumor‐targeting ligands may enhance their specificity for cancer cells overexpressing the corresponding ligand‐binding counterpart. The focus here is on anisamide, a low‐molecular‐weight benzamide derivative used as a tumor‐directing moiety in functionalized nanosystems, based on its alleged interaction with Sigma receptors. The scintigraphic agents that initially inspired the use of anisamide for tumor targeting are described, and the published anisamide‐tethered nanocarrier formulations are reviewed, together with a critical overview of the ligand's tumor‐targeting properties. Moreover, anisamide's putative but dubious cellular target, the Sigma‐1 receptor, is discussed with regard to its subcellular localization and implications in cancer. Data from in vivo studies reveal that the effect of anisamide on the antitumor efficacy of the decorated nanosystems varies considerably among the published reports. Together with the evidence questioning the interaction of anisamide with the Sigma receptors, the variability of anisamide's effect on the tumor deposition and the antitumor efficacy of the decorated drug carriers calls into question the extent of the ligand's tumor‐targeting effect. Further research is necessary to elucidate the ligand's utility in tumor targeting.     

Poly(methyl methacrylate) nanocomposites based on TiO2 nanocrystals: Tailoring material properties towards sensing

Nanocomposite materials have been obtained by dispersing organic capped TiO₂ nanocrystals (NCs) with different shape and surface chemistry in poly(methyl methacrylate) (PMMA) as a host medium. Films of the prepared nanocomposites based on TiO₂ NCs have been fabricated by spin coating and morphologically characterized as a function of the preparative conditions. The organic vapor absorption ability of the PMMA/TiO₂ NC based nanocomposites has been then investigated both for spherical and rod-like NCs, and the chemical nature of the coordinating organic molecules has been also varied. The results of the investigation have demonstrated that NC geometry and surface chemistry can modulate the specific absorption characteristics of the modified PMMA in order to absorb different solvent molecules (i.e. acetone, ethanol, propan-2-ol and water). Such features, due to specific interactions between the potential analyte vapors and the functionalized surface of NCs, can effectively be addressed in a controlled and reproducible way, thus offering original opportunities for designing innovative chemical sensors.     

Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration

To control specific endothelial cell (EC) functions, cell adhesive RGDS, EC specific REDV and YIGSR peptides, and angiogenic SVVYGLR sequences were covalently immobilized onto polyethylene terephthalate (PET) surfaces for the purpose of cell culture. X-ray photoelectron spectroscopy, atomic force microscopy, fluorescence microscopy and contact angle measurement were employed for characterization of surface modifications. The peptide density on PET surfaces was evaluated by fluorescence microscopy. The surfaces immobilized with peptides were exposed to human umbilical vein endothelial cells to study their specific effects onto EC functions. The results showed that the surface functionalized by these peptides enhanced the EC adhesion, spreading and migration as compared with native PET surfaces. Specifically, the RGDS peptides induced more cell adhesion than other peptides. The YIGSR and SVVYGLR sequences induced more cell spreading and cell migration, represented by intense focal adhesion at the leading edges of cell spreading and migration. The bi-functionalization of RGDS and SVVYGLR peptides (MIX) combined the advantages of both peptides and induced significant EC adhesion, spreading and migration. Our study indicates that the surface functionalization by peptides specific for ECs, especially the combination of RGDS with SVVYGLR or YIGSR peptides, has potential applications in promoting endothelialization of vascular prostheses and for construction of vascularized tissues in tissue engineering.     

Engineering the Surface of Smart Nanocarriers Using a pH‐/Thermal‐/GSH‐Responsive Polymer Zipper for Precise Tumor Targeting Therapy In Vivo

Nanocarrier surface chemistry plays a vital role in mediating cell internalization and enhancing delivery efficiency during in vivo chemotherapy. Inspired by the ability of proteins to alter their conformation to mediate functions, a pH‐/thermal‐/glutathione‐responsive polymer zipper consisting of cell‐penetrating poly(disulfide)s and thermosensitive polymers bearing guanidinium/phosphate (Gu⁺/pY^?) motifs to spatiotemporally tune the surface composition of nanocarriers for precise tumor targeting and efficient drug delivery is developed. Surface engineering allows the nanocarriers to remain undetected during blood circulation and favors passive accumulation at tumor sites, where the acidic microenvironment and photothermal heating break the pY^?/Gu⁺ binding and rupture the zipper, thereby exposing the penetrating shell and causing enhanced cellular uptake via counterion‐/thiol‐/receptor‐mediated endocytosis. The in vivo study demonstrates that by manipulating the surface states on command, the nanocarriers show longer blood circulation time, minimized uptake and drug leakage in normal organs, and enhanced accumulation and efficient drug release at tumor sites, greatly inhibiting tumor growth with only slight damage to normal tissues. If integrated with a photothermal dye approved by the U.S. Food and Drug Administration (FDA), polymer zipper would provide a versatile protocol for engineering nanomedicines with high selectivity and efficiency for clinical cancer treatment.     

Mechanism for the Cellular Uptake of Targeted Gold Nanorods of Defined Aspect Ratios

Biomedical applications of non‐spherical nanoparticles such as photothermal therapy and molecular imaging require their efficient intracellular delivery, yet reported details on their interactions with the cell remain inconsistent. Here, the effects of nanoparticle geometry and receptor targeting on the cellular uptake and intracellular trafficking are systematically explored by using C166 (mouse endothelial) cells and gold nanoparticles of four different aspect ratios (ARs) from 1 to 7. When coated with poly(ethylene glycol) strands, the cellular uptake of untargeted nanoparticles monotonically decreases with AR. Next, gold nanoparticles are functionalized with DNA oligonucleotides to target Class A scavenger receptors expressed by C166 cells. Intriguingly, cellular uptake is maximized at a particular AR: shorter nanorods (AR = 2) enter C166 cells more than nanospheres (AR = 1) and longer nanorods (AR = 4 or 7). Strikingly, long targeted nanorods align to the cell membrane in a near‐parallel manner followed by rotating by ≈90° to enter the cell via a caveolae‐mediated pathway. Upon cellular entry, targeted nanorods of all ARs predominantly traffic to the late endosome without progressing to the lysosome. The studies yield important materials design rules for drug delivery carriers based on targeted, anisotropic nanoparticles.