Engineered Proteinticles for Targeted Delivery of siRNA to Cancer Cells

Considering the problems of small interfering RNA (siRNA) delivery using traditional viral and nonviral vehicles, a new siRNA delivery system to enhance efficiency and safety needs to be developed. Here human ferritin‐based proteinticles are genetically engineered to simultaneously display various functional peptides on the surface of proteinticles: cationic peptide to capture siRNA, tumor cell targeting and penetrating peptides, and enzymatically cleaved peptide to release siRNA inside tumor cell. In the in vitro treatment of poly‐siRNA‐proteinticle complex, both of the tumor cell targeting and penetrating peptides are important for efficient delivery of siRNA, and the red fluorescent protein (RFP) expression in RFP‐expressing tumor cells is notably suppressed by the delivered siRNA with the complementary sequence to RFP mRNA. It seems that the human ferritin‐based proteinticle is an efficient, stable, and safe tool for siRNA delivery, having a great potential for application to in vivo cancer treatment. The unique feature of proteinticles is that multiple and functional peptides can be simultaneously and evenly placed and also easily switched on the proteinticle surface through a simple genetic modification, which is likely to make proteinticles appropriate for targeted delivery of siRNA to a wide range of cancer cells.     

Bioengineered Extracellular Membranous Nanovesicles for Efficient Small‐Interfering RNA Delivery: Versatile Platforms for Stem Cell Engineering and In Vivo Delivery

Naturally derived nanovesicles secreted from various cell types and found in body fluids can provide effective platforms for the delivery of various cargoes because of their intrinsic ability to be internalized for intercellular signal transmission and membrane recycling. In this study, the versatility of bioengineered extracellular membranous nanovesicles as potent carriers of small‐interfering RNAs (siRNAs) for stem cell engineering and in vivo delivery has been explored. Here, exosomes have been engineered, one of the cell‐derived vesicle types, to overexpress exosomal proteins fused with cell‐adhesion or cell‐penetrating peptides for enhanced intracellular gene transfer. To devise a more effective delivery system with potential for mass production, a new siRNA delivery system has also been developed by artificially inducing the outward budding of plasma membrane nanovesicles. Those nanovesicles have been engineered by overexpressing E‐cadherin to facilitate siRNA delivery to human stem cells with resistance to intracellular gene transfer. Both types of engineered nanovesicles deliver siRNAs to human stem cells for lineage specification with negligible cytotoxicity. The nanovesicles are efficient in delivering siRNA in vivo, suggesting feasibility for gene therapy. Cell‐derived, bioengineered nanovesicles used for siRNA delivery can provide functional platforms enabling effective stem cell therapeutics and in vivo gene therapy.     

Properties of Native High‐Density Lipoproteins Inspire Synthesis of Actively Targeted In Vivo siRNA Delivery Vehicles

Efficient systemic administration of therapeutic short interfering RNA (siRNA) is challenging. High‐density lipoproteins (HDLs) are natural in vivo RNA delivery vehicles. Specifically, native HDLs: 1) load single‐stranded RNA; 2) are anionic, which requires charge reconciliation between the RNA and HDL, and 3) actively target scavenger receptor type B‐1 (SR‐B1) to deliver RNA. Emphasizing these particular parameters, templated lipoprotein particles (TLP), mimics of spherical HDLs, are employed and are self‐assembled with single‐stranded complements of, presumably, any highly unmodified siRNA duplex pair after formulation with a cationic lipid. Resulting siRNA templated lipoprotein particles (siRNA‐TLP) are anionic and tunable with regard to RNA assembly and function. Data demonstrate that the siRNA‐TLPs actively target SR‐B1 to potently reduce androgen receptor and enhancer of zeste homolog 2 proteins in multiple cancer cell lines. Systemic administration of siRNA‐TLPs demonstrated no off‐target toxicity and significantly reduced the growth of prostate cancer xenografts. Thus, native HDLs inspired the synthesis of a hybrid siRNA delivery vehicle that can modularly load single‐stranded RNA complements after charge reconciliation with a cationic lipid, and that function due to active targeting of SR‐B1.     

Structure‐Invertible Nanoparticles for Triggered Co‐Delivery of Nucleic Acids and Hydrophobic Drugs for Combination Cancer Therapy

Here, a new type of structure‐invertible, redox‐responsive polymeric nanoparticle for the efficient co‐delivery of nucleic acids and hydrophobic drugs in vitro and in vivo is reported for the first time, to combat the major challenges facing combination cancer therapy. The co‐delivery vector, which is prepared by conjugating branched poly(ethylene glycol) with dendrimers of two generations (G2) through disulfide linkages, is able to complex nucleic acids and load hydrophobic drugs with high loading capacity through structure inversion. The cleavage of disulfide linkages at intracellular glutathione‐rich reduction environment significantly decreases the cytotoxicity, and promotes more efficient drug release and gene transfection in vitro and in vivo. The co‐delivery carrier also displays enhanced endosomal escape capability and improved serum stability in vitro as compared with G2, and exhibits prolonged residence time and stronger transfection activity in vivo. Most importantly, co‐delivery of doxorubicin (DOX) and B‐cell lymphoma 2 (Bcl‐2) small interfering RNA (siRNA) exerts a combinational effect against tumor growth in murine tumor models in vivo, which is much more effective than either DOX or Bcl‐2 siRNA‐based monotherapy. The structure‐invertible nanoparticles may constitute a promising stimuli‐responsive system for the efficacious co‐delivery of multiple cargoes in future clinical applications of combination cancer therapies.     

Lipid‐Like Nanoparticles for Small Interfering RNA Delivery to Endothelial Cells

Here, nanoparticles composed of lipid‐like materials (lipidoids) to facilitate non‐viral delivery of small interfering RNA (siRNA) to endothelial cells (ECs) are developed. Nanoparticles composed of siRNA and lipidoids with small size (～200 nm) and positive charge (～34 mV) are formed by self‐assembly of lipidoids and siRNA. Ten lipidoids are synthesized and screened for their ability to facilitate the delivery of siRNA into ECs. Particles composed of leading lipidoids show significantly better delivery to ECs than a leading commercially available transfection reagent, Lipofectamine 2000. As a model of potential therapeutic application, nanoparticles composed of the top performing lipidoid, NA114, are studied for their ability to deliver siRNA targeting anti‐angiogenic factor (SHP‐1) to human ECs. Silencing of SHP‐1 expression significantly enhances EC proliferation and decreases EC apoptosis under a simulated ischemic condition.     

Optimizing the Size of Micellar Nanoparticles for Efficient siRNA Delivery

Delivery of small interfering RNA (siRNA) by nanocarriers has shown promising therapeutic potential in cancer therapy. However, poor understanding of the correlation between the physicochemical properties of nanocarriers and their interactions with biological systems has significantly hindered its anticancer efficacy. Herein, in order to identify the optimal size of nanocarriers for siRNA delivery, different sized cationic micellar nanoparticles (MNPs) (40, 90, 130, and 180 nm) are developed that exhibit similar siRNA binding efficacies, shapes, surface charges, and surface chemistries (PEGylation) to ensure size is the only variable. Size‐dependent biological effects are carefully and comprehensively evaluated through both in vitro and in vivo experiments. Among these nanocarriers, the 90 nm MNPs show the optimal balance of prolonged circulation and cellular uptake by tumor cells, which result in the highest retention in tumor cells. In contrast, larger MNPs are rapidly cleared from the circulation and smaller MNPs are inefficiently taken up by tumor cells. Accordingly, 90 nm MNPs carrying polo‐like kinase 1 (Plk1)‐specific siRNA (siPlk1) show superior antitumor efficacy, indicating that 90 nm could either be the optimal size for systemic delivery of siRNA or close to it. Our findings provide valuable information for rationally designing nanocarriers for siRNA‐based cancer therapy in the future.     

Nanogel-Facilitated Protein Intracellular Specific Degradation through Trim-Away

The recently discovered “Trim-Away” mechanism opens a new window for fast and selective degradation of endogenous proteins. However, the in vivo and clinical application of this approach is hindered by the requirement of special skills and equipment needed for the intracellular delivery of antibodies. Here, an antibody conjugated polymer nanogel system, nano gel-facilitate d pr otein intra cellular s pecific de gr adation (Nano-ERASER), for intracellular delivery and release of antibody, and degradation of a specific endogenous protein is developed. After being delivered into cells, the antibody is released and forms a complex with its target protein, and subsequently binds to the Fc receptor of Tripartite motif 21 (TRIM21). The resulting complex of target protein/antibody/TRIM21 is then degraded by the proteasome. The efficacy of Nano-ERASER is validated by depleting GFP protein in a GFP expressing cell line. Furthermore, Nano-ERASER successfully degrades the coatomer protein complex ζ1, a vital protein for cancer cells, and kills those cells while sparing normal cells. Benefitting from its convenience and targeted delivery merit, Nano-ERASER technique is promising in providing a reliable tool for endogenous protein function study as well as paves the way for novel antibody-based Trim-Away therapeutic modalities for cancer and other diseases.     

Matrix Metalloproteinase Responsive,Proximity‐Activated Polymeric Nanoparticles for siRNA Delivery

Small interfering RNA (siRNA) has significant potential to evolve into a new class of pharmaceutical inhibitors, but technologies that enable robust, tissue‐specific intracellular delivery must be developed before effective clinical translation can be achieved. A pH‐responsive, smart polymeric nanoparticle (SPN) with matrix metalloproteinase (MMP)‐7‐dependent proximity‐activated targeting (PAT) is described here. The PAT‐SPN is designed to trigger cellular uptake and cytosolic delivery of siRNA once activated by MMP‐7, an enzyme whose overexpression is a hallmark of cancer initiation and progression. The PAT‐SPN is composed of a corona‐forming polyethylene glycol (PEG) block, an MMP‐7‐cleavable peptide, a cationic siRNA‐condensing block, and a pH‐responsive, endosomolytic terpolymer block that drives self‐assembly and forms the PAT‐SPN core. With this novel design, the PEG corona shields cellular interactions until it is cleaved in MMP‐7‐rich environments, shifting the SPN ζ‐potential from +5.8 to +14.4 mV and triggering a 2.5 fold increase in carrier internalization. The PAT‐SPN exhibits pH‐dependent membrane disruptive behavior that enables siRNA escape from endo‐lysosomal pathways. Intracellular siRNA delivery and knockdown of the model enzyme luciferase in R221A‐Luc mammary tumor cells is significantly increased by MMP‐7 pre‐activation (p < 0.05). These combined data indicate that the PAT‐SPN provides a promising new platform for tissue‐specific, proximity‐activated siRNA delivery to MMP‐rich pathological environments.     

Triple Enzyme-Regulated Molecular Hydrogels for Carrier-Free Delivery of Lonidamine

Cancerous cells exhibit overexpression of multiple enzymes in various cellular compartments. These enzymes are often undruggable, yet display unique advantages in regulating intracellular self-assembly and dis-assembly of small molecules for cancer targeting. Herein, a self-assembling molecule (LND-1p-ES) for carrier-free delivery of lonidamine specifically to cancerous cells is designed, where LND-1p-ES executes as both a drug and a carrier with combined benefits thereof. Under the precise regulation of phosphatase (ALP) and esterase (CES), LND-1p-ES is capable of self-assembling intracellularly in a spatiotemporal manner, to confer lonidamine-borne nanofibers with enhanced cellular uptake. These nanofibers also facilitate controlled drug release with the aid of cellular proteases. Taking advantages of overexpressing ALP and CES as well as proteases in cancerous cells, the LND-1p-ES formulation demonstrates enhanced potency and selectivity against melanoma cells A375 in vitro and in vivo. In comparison, none/single enzyme responsive compounds fail to show a similar potency or selectivity, further confirming the indispensable roles of these enzymes in the delivery system. Collectively, the research provides a viable strategy to utilize multiple enzymes in cancerous cells for regulation of intracellular self-assembly, which can be expanded to design smart soft materials responsive to multiple biologically relevant biomolecules for enhanced therapeutic efficacy.     

Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery

The successful therapeutic application of small interfering RNA (siRNA) largely relies on the development of safe and effective delivery systems that are able to guide the siRNA therapeutics to the cytoplasm of the target cell. In this report, biodegradable cationic dextran nanogels are engineered by inverse emulsion photopolymerization and their potential as siRNA carriers is evaluated. The nanogels are able to entrap siRNA with a high loading capacity, based on electrostatic interaction. Confocal microscopy and flow cytometry analysis reveal that large amounts of siRNA‐loaded nanogels can be internalized by HuH‐7 human hepatoma cells without significant cytotoxicity. Following their cellular uptake, it is found that the nanogels are mainly trafficked towards the endolysosomes. The influence of two different strategies to enhance endosomal escape on the extent of gene silencing is investigated. It is found that both the application of photochemical internalization (PCI) and the use of an influenza‐derived fusogenic peptide (diINF‐7) can significantly improve the silencing efficiency of siRNA‐loaded nanogels. Furthermore, it is shown that an efficient gene silencing requires the degradation of the nanogels. As the degradation kinetics of the nanogels can easily be tailored, these particles show potential for intracellular controlled release of short interfering RNA.     

Enhanced Intracellular Protein Transduction by Sequence Defined Tetra‐Oleoyl Oligoaminoamides Targeted for Cancer Therapy

Intracellular protein delivery presents a novel promising prospect for cell biology research and cancer therapy. However, inefficient cellular uptake and lysosomal sequestration hinder productive protein delivery into the cytosol. Here, a library of 16 preselected sequence‐defined oligoaminoamide oligomers is evaluated for intracellular protein delivery. All oligomers, containing polyethylene glycol (PEG) for shielding and optionally folic acid as targeting ligand, manifest cellular internalization of disulfide‐conjugated enhanced green fluorescent protein (EGFP). However, only a PEGylated folate‐receptor targeted two‐arm oligomer (729) containing both arms terminally modified with two oleic acids shows persistent intracellular protein survival and nuclear import of nlsEGFP (which contains a nuclear localization sequence) in folate‐receptor‐positive KB carcinoma cells, validating both effective endolysosomal escape and following subcellular transport. Furthermore, using ribonuclease A as a therapeutic cargo protein, among the tested oligomers, the oleic acid modified targeted two‐arm oligomers exert the most significant tumor cell killing of KB tumor cells. An investigation of structure–activity relationship elucidates that the incorporated oleic acids play a vital role in the enhanced intracellular protein delivery, by promoting stable formation of 25–35 nm lipo‐oligomer protein nanoparticles and by membrane‐active characteristics facilitating intracellular cytosolic delivery.     

A Charge Reversible Self‐Delivery Chimeric Peptide with Cell Membrane‐Targeting Properties for Enhanced Photodynamic Therapy

The cell membrane is the most important protective barrier in living cells and cell membrane targeted therapy may be a high‐performance therapeutic modality for tumor treatment. Here, a novel charge reversible self‐delivery chimeric peptide C₁₆–PRP–DMA is developed for long‐term cell membrane targeted photodynamic therapy (PDT). The self‐assembled C₁₆–PRP–DMA nanoparticles can effectively target to tumor by enhanced permeability and retention effect without additional carriers. After undergoing charge reverse in acidic tumor microenvironment, C₁₆–PRP–DMA inserts into the tumor cell membrane with a long retention time of more than 14 h, which is very helpful for in vivo applications. It is found that under light irradiation, the reactive oxygen species generated by the inserted C₁₆–PRP–DMA would directly disrupt cell membrane and rapidly induce cell necrosis, which remarkably increases the PDT effect in vitro and in vivo. This novel self‐delivery chimeric peptide with a long‐term cell membrane targeting property provides a new prospect for effective PDT of cancer.     

Self‐Assembly of Therapeutic Peptide into Stimuli‐Responsive Clustered Nanohybrids for Cancer‐Targeted Therapy

Clinical translation of therapeutic peptides, particularly those targeting intracellular protein–protein interactions (PPIs), has been hampered by their inefficacious cellular internalization in diseased tissue. Therapeutic peptides engineered into nanostructures with stable spatial architectures and smart disease targeting ability may provide a viable strategy to overcome the pharmaceutical obstacles of peptides. This study describes a strategy to assemble therapeutic peptides into a stable peptide–Au nanohybrid, followed by further self‐assembling into higher‐order nanoclusters with responsiveness to tumor microenvironment. As a proof of concept, an anticancer peptide termed β‐catenin/Bcl9 inhibitors is copolymerized with gold ion and assembled into a cluster of nanohybrids (pCluster). Through a battery of in vitro and in vivo tests, it is demonstrated that pClusters potently inhibit tumor growth and metastasis in several animal models through the impairment of the Wnt/β‐catenin pathway, while maintaining a highly favorable biosafety profile. In addition, it is also found that pClusters synergize with the PD1/PD‐L1 checkpoint blockade immunotherapy. This new strategy of peptide delivery will likely have a broad impact on the development of peptide‐derived therapeutic nanomedicine and reinvigorate efforts to discover peptide drugs that target intracellular PPIs in a great variety of human diseases, including cancer.     

Dual‐Drug Delivery Using Dextran‐Functionalized Nanoparticles Targeting Cardiac Fibroblasts for Cellular Reprogramming

The inability of the heart to recover from an ischemic insult leads to the formation of fibrotic scar tissue and heart failure. From the therapeutic strategies under investigation, cardiac regeneration holds the promise of restoring the full functionality of a damaged heart. Taking into consideration the presence of vast numbers of fibroblasts and myofibroblasts in the injured heart, direct fibroblast reprogramming into cardiomyocytes using small drug molecules is an attractive therapeutic option to replenish the lost cardiomyocytes. Here, a spermine‐acetalated dextran‐based functional nanoparticle is developed for pH‐triggered drug delivery of two poorly water soluble small molecules, CHIR99021 and SB431542, both capable of increasing the efficiency of direct reprogramming of fibroblast into cardiomyocytes. Upon functionalization with polyethylene glycol and atrial natriuretic peptide, the biocompatibility of the nanosystem is improved, and the cellular interactions with the cardiac nonmyocytes are specifically augmented. The dual delivery of the compounds is verified in vitro, and the compounds exerted concomitantly anticipate biological effects by stabilizing β‐catenin (CHIR99021) and by preventing translocation of Smad3 to the nucleus of (myo)fibroblasts (SB431542). These observations highlight the potential of this nanoparticle‐based system toward improved drug delivery and efficient direct reprogramming of fibroblasts into cardiomyocyte‐like cells, and thus, potential cardiac regeneration therapy.     

A Fluorinated Bola‐Amphiphilic Dendrimer for On‐Demand Delivery of siRNA,via Specific Response to Reactive Oxygen Species

Functional materials capable of responding to stimuli intrinsic to diseases are extremely important for specific drug delivery at the disease site. However, developing on‐demand stimulus‐responsive vectors for targeted delivery is highly challenging. Here, a stimulus‐responsive fluorinated bola‐amphiphilic dendrimer is reported for on‐demand delivery of small interfering RNA (siRNA) in response to the characteristic high level of reactive oxygen species (ROS) in cancer cells. This dendrimer bears a ROS‐sensitive thioacetal in the hydrophobic core and positively charged poly(amidoamine) dendrons at the terminals, capable of interacting and compacting the negatively charged siRNA into nanoparticles to protect the siRNA and promote cellular uptake. The ROS‐sensitive feature of this dendrimer boosts specific and efficient disassembly of the siRNA/vector complexes in ROS‐rich cancer cells for effective siRNA delivery and gene silencing. Moreover, the fluorine tags in the vector enable ¹⁹F‐NMR analysis of the ROS‐responsive delivery process. In addition, this ingenious and distinct bola‐amphiphilic dendrimer is also able to combine the advantageous delivery features of both lipid and dendrimer vectors. Therefore, it represents an innovative on‐demand stimulus‐responsive delivery platform.     

Photocrosslinked Bioreducible Polymeric Nanoparticles for Enhanced Systemic siRNA Delivery as Cancer Therapy

Clinical translation of polymer-based nanocarriers for systemic delivery of RNA has been limited due to poor colloidal stability in the blood stream and intracellular delivery of the RNA to the cytosol. To address these limitations, this study reports a new strategy incorporating photocrosslinking of bioreducible nanoparticles for improved stability extracellularly and rapid release of RNA intracellularly. In this design, the polymeric nanocarriers contain ester bonds for hydrolytic degradation and disulfide bonds for environmentally triggered small interfering RNA (siRNA) release in the cytosol. These photocrosslinked bioreducible nanoparticles (XbNPs) have a shielded surface charge, reduced adsorption of serum proteins, and enable superior siRNA-mediated knockdown in both glioma and melanoma cells in high-serum conditions compared to non-crosslinked formulations. Mechanistically, XbNPs promote cellular uptake and the presence of secondary and tertiary amines enables efficient endosomal escape. Following systemic administration, XbNPs facilitate targeting of cancer cells and tissue-mediated siRNA delivery beyond the liver, unlike conventional nanoparticle-based delivery. These attributes of XbNPs facilitate robust siRNA-mediated knockdown in vivo in melanoma tumors colonized in the lungs following systemic administration. Thus, biodegradable polymeric nanoparticles, via photocrosslinking, demonstrate extended colloidal stability and efficient delivery of RNA therapeutics under physiological conditions, and thereby potentially advance systemic delivery technologies for nucleic acid-based therapeutics.     

Pro-Peptide-Reinforced,Mucus-Penetrating Pulmonary siRNA Delivery Mitigates Cytokine Storm in Pneumonia

Pulmonary delivery of anti-inflammatory siRNA holds great potential in mitigating the cytokine storm during severe pneumonia. However, commonly utilized polycationic siRNA delivery vehicles can hardly penetrate the mucus barrier, thus greatly hurdling their therapeutic efficacy. Herein, TNF-α siRNA (siTNF-α) delivery nanocomplexes (NCs) are engineered with mucus/cytomembrane dual-penetration capabilities, realized via surface-coating of NCs with RC, an inflammation-sheddable, charge-reversal pro-peptide of RAGE-binding peptide (RBP). RC-coated dendritic poly-_L-lysine/siTNF-α (DsT) NCs possess negative surface charges, and can thus efficiently penetrate the mucus layer after intratracheal administration. In the inflamed alveolar space with mild acidity, RC recovers to the cationic RBP and shed off, re-exposing the DsT NCs that efficiently transfect the alveolar macrophages and provokes TNF-α silencing. Thus, siTNF-α and RBP cooperatively alleviate the uncontrolled inflammation during acute lung injury. This study renders a unique approach for mediating trans-mucus nucleic acid delivery, and will find promising utilities for the treatment of severe pneumonia.     

Ribonucleoprotein: A Biomimetic Platform for Targeted siRNA Delivery

Many small interfering RNA (siRNA) carriers have been developed over the years, mostly based on cationic lipids and polymers that can condense siRNA into nanoparticle complexes and aid in endosomal escape. In comparison, development of charge‐neutral siRNA carriers to avoid or reduce nonspecific binding, aggregation, and toxicity is limited, due to a lack of mechanisms for carrier‐siRNA association beyond electrostatic interaction. Here, a unique, charge‐neutral, biomimetic platform is reported, mimicking the natural state of functional RNA, ribonucleoprotein (RNP). This RNP architecture is simple to make, precise in assembly stoichiometry, stable in serum, and biocompatible. Gene knockdown is achieved in vitro and in vivo, demonstrating excellent potential for translation.     

Encapsulating a Hydrophilic Chemotherapeutic into Rod‐Like Nanoparticles of a Genetically Encoded Asymmetric Triblock Polypeptide Improves Its Efficacy

Encapsulating hydrophilic chemotherapeutics into the core of polymeric nanoparticles can improve their therapeutic efficacy by increasing their plasma half‐life, tumor accumulation, and intracellular uptake, and by protecting them from premature degradation. To achieve these goals, a recombinant asymmetric triblock polypeptide (ATBP) that self‐assembles into rod‐shaped nanoparticles, and which can be used to conjugate diverse hydrophilic molecules, including chemotherapeutics, into their core is designed. These ATBPs consist of three segments: a biodegradable elastin‐like polypeptide, a hydrophobic tyrosine‐rich segment, and a short cysteine‐rich segment, that spontaneously self‐assemble into rod‐shaped micelles. Covalent conjugation of a structurally diverse set of hydrophilic small molecules, including a hydrophilic chemotherapeutic—gemcitabine—to the cysteine residues also leads to formation of nanoparticles over a range of ATBP concentrations. Gemcitabine‐loaded ATBP nanoparticles have significantly better tumor regression compared to free drug in a murine cancer model. This simple strategy of encapsulation of hydrophilic small molecules by conjugation to an ATBP can be used to effectively deliver a range of water‐soluble drugs and imaging agents in vivo.     

An Integrated Therapeutic Delivery System for Enhanced Treatment of Hepatocellular Carcinoma

Nanomaterials hold promise for the treatment of human carcinomas but integrating multiple functions into a single drug carrier system remains challenging. Herein, an integrated therapeutic delivery system for human hepatocellular carcinoma (HCC) treatment is reported, which is based on rhodamine B (RhB) end‐labeled cationic poly[2‐(dimethylamino)ethyl methacrylate] (PDMAEMA) and hydrophobic poly(3‐azido‐2‐hydroxypropyl methacrylate) (PGMA‐N₃) segments equipped with a covalently bound galactose. This biocompatible and safe platform RhB‐PDMAEMA25‐c‐PGMA50‐Gal micelles (Gal‐micelles) offers four advantages: (1) Galactose ligands enhance cellular uptake by targeting the asialoglycoprotein receptor (ASGPR) that is overexpressed on HCC cell lines surfaces; (2) RhB end‐labeling facilitates real‐time imaging for tracking both in vitro and in vivo; (3) the acidic tumor microenvironment protonates the carrier system for efficient drug release as well as gene transfection, (4) codelivery of anticancer drug doxorubicin (DOX) and B‐cell lymphoma 2 small interfering RNA (Bcl‐2 siRNA) works synergistically against tumor growth in both subcutaneous and orthotopic HCC bearing mouse models. This integrated therapeutic delivery system holds potential for future clinical HCC treatment.