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RGD-Modified Apoferritin Nanoparticles for Efficient Drug Delivery to Tumors
Abstract
Ferritin is a major iron storage protein found in humans and most living organisms. Each ferritin is comprised of 24 subunits, which self-assemble to form a cage-like nanostructure. FRT nanocages can be genetically modified to present a peptide sequence on the surface. Recently, we demonstrated that Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (RGD4C)-modified ferritin can efficiently home to tumors through RGD-integrin αvβ3 interaction. Though promising, studies on evaluating surface modified ferritin nanocages as drug delivery vehicles have seldom been reported. Herein we showed that after being pre-complexed with Cu(II), doxorubicin can be loaded onto RGD modified apoferritin nanocages with high efficiency (up to 73.49wt%). When studied on U87MG subcutaneous tumor models, these doxorubicin-loaded ferritin nanocages showed a longer circulation half-life, higher tumor uptake, better tumor growth inhibition, and less cardiotoxicity than free doxorubicin. Such a technology might be extended to load a broad range of therapeutics and holds great potential in clinical translation.
... Key biomedical applications of NVPNs include drug delivery, vaccine development and diagnostic bioimaging (Lai et al., 2009;Suci et al., 2010;Ren et al., 2012;Jeon et al., 2013;Toita et al., 2013;Zhen et al., 2013;Moon et al., 2014;Ra et al., 2014;Lee E. J. et al., 2015;Lee W.et al., 2015). Non-natural, bioinspired NVPNs can also be designed de novo through the assembly of artificial, functional monomers (Vázquez and Villaverde, 2010;Schreiber and Schiller, 2013;Bhaskar and Lim, 2017;Diaz et al., 2018). ...
... Next, cells are resuspended in an appropriate buffer, and the intracellular nanocages are released via a specific cell lysis step. The most common options at lab scale are ultrasonication (Allen et al., 2002;Suci et al., 2010;Choi et al., 2011;Jeon et al., 2013;Zhen et al., 2013;Bellini et al., 2014;Cassidy-Amstutz et al., 2016;Martín et al., 2022), French press (Jung et al., 2002;Suci et al., 2010;Peng and Lim, 2011) and Dounce homogenization cell lysis (Stephen et al., 2001;Poderycki et al., 2006). In some instances, lysozyme is added to the lysis buffer to weaken/break down bacterial cell walls and hence improve lysis (Allen et al., 2002;Suci et al., 2010;Bellini et al., 2014). ...
... Thus, heating clarified E. coli cell lysates at 65°C-90°C will promote the denaturation and precipitation of host proteins, which are subsequently removed by centrifugation (e.g., 12,000 × g), without affecting the structure of the thermostable nanocages . This strategy was applied successfully to lysates containing a range of different nanocages, including Dps (Allen et al., 2002;Suci et al., 2010), E2 nanocages (Dalmau et al., 2008;2009), encapsulin (Moon et al., 2014;Cassidy-Amstutz et al., 2016), ferritin (Santambrogio et al., 2000;Sana et al., 2010;Falvo et al., 2013;Zhen et al., 2013;Bellini et al., 2014;He and Marles-Wright, 2015), sHSP (Flenniken et al., 2006) and I3-01 nanocages . ...
Protein nanocages are highly ordered nanometer scale architectures, which are typically formed by homo- or hetero-self-assembly of multiple monomers into symmetric structures of different size and shape. The intrinsic characteristics of protein nanocages make them very attractive and promising as a biological nanomaterial. These include, among others, a high surface/volume ratio, multi-functionality, ease to modify or manipulate genetically or chemically, high stability, mono-dispersity, and biocompatibility. Since the beginning of the investigation into protein nanocages, several applications were conceived in a variety of areas such as drug delivery, vaccine development, bioimaging, biomineralization, nanomaterial synthesis and biocatalysis. The ability to generate large amounts of pure and well-folded protein assemblies is one of the keys to transform nanocages into clinically valuable products and move biomedical applications forward. This calls for the development of more efficient biomanufacturing processes and for the setting up of analytical techniques adequate for the quality control and characterization of the biological function and structure of nanocages. This review concisely covers and overviews the progress made since the emergence of protein nanocages as a new, next-generation class of biologics. A brief outline of non-viral protein nanocages is followed by a presentation of their main applications in the areas of bioengineering, biotechnology, and biomedicine. Afterwards, we focus on a description of the current processes used in the manufacturing of protein nanocages with particular emphasis on the most relevant aspects of production and purification. The state-of-the-art on current characterization techniques is then described and future alternative or complementary approaches in development are also discussed. Finally, a critical analysis of the limitations and drawbacks of the current manufacturing strategies is presented, alongside with the identification of the major challenges and bottlenecks.
... As the TFRC ligand [18], ferritin is a naturally spherical iron storage protein comprising 24 self-assembled subunits of two types, the heavy (H) and light (L) chains, with a ratio that varies drastically [15,19,20]. Ferritin protein self-assembles spontaneously in the physiological environment into a hollow nanocage with an outer diameter of 12 nm and an interior cavity with a diameter of 8 nm [21], thus allowing the rapid expression of functional ferritin in prokaryotic systems [22]. ...
... Ferritin protein self-assembles spontaneously in the physiological environment into a hollow nanocage with an outer diameter of 12 nm and an interior cavity with a diameter of 8 nm [21], thus allowing the rapid expression of functional ferritin in prokaryotic systems [22]. The inner cavity could provide potential space to encapsulate small molecule drugs as well as offer protection from the degradation of drug molecules [15,17,19,20]. In vivo, the ferritin H chain can be recognized by TFRC and taken up by cells [18]. ...
... In vivo, the ferritin H chain can be recognized by TFRC and taken up by cells [18]. Unlike conventional synthetic or modified materials, ferritin is a natural protein in the body and therefore is biocompatible and nonimmunogenic [19,20]. In addition, despite their rigidity under physiological conditions, the ferritin nanocages can disassemble into subunits in an acidic environment (e.g., pH = 2) and be reconstituted into a nanocage structure when the pH returns to neutral (e.g., pH = 7.4) [23][24][25]. ...
Background
Currently, high doses of cytarabine arabinoside (Ara-C)-based combined chemotherapy are commonly used in acute myeloid leukemia (AML) therapy, but severe adverse effects and poor suppression effects in leukemia cells limit the clinical therapeutic efficiency of Ara-C-based chemotherapy due to a lack of targeting selectivity. To improve the therapeutic effect of Ara-C in AML, here, since we confirmed that transferrin receptor 1 (TFRC) expression in AML cells was constant, we generated Ara-C@HFn by encapsulating free Ara-C into self-assembled heavy ferritin chain (HFn, the ligand of TFRC) nanocages.
Results
The analysis of clinically relevant data suggested that the high expression levels of TFRC from AML cells would not decrease significantly after treatment with Ara-C. Ara-C@HFn can be efficiently internalized by leukemia cells, showing stronger cytotoxic effects in vitro and reducing the burden of leukemia in AML mice more effectively in vivo than free Ara-C. Ara-C@HFn treatment showed no acute toxicity in visceral organs of mice. Moreover, the analysis of clinically relevant data also suggested that there are several drugs (such as tamibarotene and ABT199) that would not cause significant expression down-regulation of TFRC in AML cells (after treatment).
Conclusion
The above results suggested that TFRC can be used as a constant and effective target for drug targeting delivery of AML cells. Thus Ara-C@HFn treatment can become a safe and efficient strategy for AML therapy by specifically delivering Ara-C to AML cells. Besides, the HFn nanocages are promising for improving antineoplastic effect of other AML-related therapy drugs that do not cause downregulated expression of TFRC in AML cells.
Graphical Abstract
... In addition, as a self-assembled protein, ferritin possesses various properties that render it suitable for use as a drug nanocarrier, including thermal stability (tolerance of high temperatures of 80-100 °C), pH stability (pH 3-10), monodispersity, and biodegradability [28]. In addition, artificial modification of ferritin can further improve the targeting of ferritin carriers, which substantially improves their safety characteristics [29,30]. Engineered ferritin nanocages can target cancer-associated fibroblasts in tumors, allowing for greater precision in cancer treatment [31]. ...
... In addition, as a self-assembled protein, ferritin possesses various properties that render it suitable for use as a drug nanocarrier, including thermal stability (tolerance of high temperatures of 80-100 • C), pH stability (pH 3-10), monodispersity, and biodegradability [28]. In addition, artificial modification of ferritin can further improve the targeting of ferritin carriers, which substantially improves their safety characteristics [29,30]. Engineered ferritin nanocages can target cancer-associated fibroblasts in tumors, allowing for greater precision in cancer treatment [31]. ...
Ongoing research is gradually broadening the idea of cancer treatment, with attention being focused on nanoparticles to improve the stability, therapeutic efficacy, targeting, and other important metrics of conventional drugs and traditional drug delivery methods. Studies have demonstrated that drug delivery carriers based on biomaterials (e.g., protein nanoparticles and lipids) and inorganic materials (e.g., metal nanoparticles) have potential anticancer effects. Among these carriers, self-assembled proteins and peptides, which are highly biocompatible and easy to standardize and produce, are strong candidates for the preparation of anticancer drugs. Breast cancer (BC) and cervical cancer (CC) are two of the most common and deadly cancers in women. These cancers not only threaten lives globally but also put a heavy burden on the healthcare system. Despite advances in medical care, the incidence of these two cancers, particularly CC, which is almost entirely preventable, continues to rise, and the mortality rate remains steady. Therefore, there is still a need for in-depth research on these two cancers to develop more targeted, efficacious, and safe therapies. This paper reviews the types of self-assembling proteins and peptides (e.g., ferritin, albumin, and virus-like particles) and natural products (e.g., soy and paclitaxel) commonly used in the treatment of BC and CC and describes the types of drugs that can be delivered using self-assembling proteins and peptides as carriers (e.g., siRNAs, DNA, plasmids, and mRNAs). The mechanisms (including self-assembly) by which the natural products act on CC and BC are discussed. The mechanism of action of natural products on CC and BC and the mechanism of action of self-assembled proteins and peptides have many similarities (e.g., NF-KB and Wnt). Thus, natural products using self-assembled proteins and peptides as carriers show potential for the treatment of BC and CC.
... The strategic design of protein cages has resulted in the creation of biomolecular platforms capable of encapsulating a diverse set of guest molecules ranging from drugs [8,19], metal nanoparticles [20,21], enzymes [22,23], polymers [24,25], and oligonucleotides [26]. Applications of such systems to the areas of electrical device fabrication [27][28][29], tissue and cellular imaging [30,31], drug delivery [32,33], and metal nanoparticle generation [34] have been reported. Research on nanodimensional protein cages continues to generate more complex macro-scale arrangements, which will inevitably lead to additional applications [35][36][37]. ...
... Further molecular design principles can be anticipated from these proof-of-principle findings, which provide a unique nanodimensional tool for biomaterials applications. Future studies will focus on developing alternate cavity affinity strategies as well as utilizing N-terminal fusion peptides and chemical modification to functionalize the Bfr outer surface for targeting applications [32,62,65,126]. ...
Currently, intense interest is focused on the discovery and application of new multisubunit cage proteins and spherical virus capsids to the fields of bionanotechnology, drug delivery, and diagnostic imaging as their internal cavities can serve as hosts for fluorophores or bioactive molecular cargo. Bacterioferritin is unusual in the ferritin protein superfamily of iron-storage cage proteins in that it contains twelve heme cofactors and is homomeric. The goal of the present study is to expand the capabilities of ferritins by developing new approaches to molecular cargo encapsulation employing bacterioferritin. Two strategies were explored to control the encapsulation of a diverse range of molecular guests compared to random entrapment, a predominant strategy employed in this area. The first was the inclusion of histidine-tag peptide fusion sequences within the internal cavity of bacterioferritin. This approach allowed for the successful and controlled encapsulation of a fluorescent dye, a protein (fluorescently labeled streptavidin), or a 5 nm gold nanoparticle. The second strategy, termed the heme-dependent cassette strategy, involved the substitution of the native heme with heme analogs attached to (i) fluorescent dyes or (ii) nickel-nitrilotriacetate (NTA) groups (which allowed for controllable encapsulation of a histidine-tagged green fluorescent protein). An in silico docking approach identified several small molecules able to replace the heme and capable of controlling the quaternary structure of the protein. A transglutaminase-based chemoenzymatic approach to surface modification of this cage protein was also accomplished, allowing for future nanoparticle targeting. This research presents novel strategies to control a diverse set of molecular encapsulations and adds a further level of sophistication to internal protein cavity engineering.
... Ferritins (Fts) are ubiquitous proteins in nature and very versatile systems for biotechnology applications. The human heavy chain ferritin (hHFt) nanocages are ideal for the delivery of anticancer drugs due to their lack of immunogenicity and selective interaction with tumor cells (Li et al., 2010;Zhen et al., 2013). However, the current protocols for disassembling and reassembling hHFt to load cargo molecules have still some drawbacks, such as low protein recovery (Zhang et al., 2020;Zhang et al., 2021) and homogeneity. ...
... Ferritins (Fts) are ubiquitous proteins in nature and very versatile systems for biotechnology applications. The human heavy chain ferritin (hHFt) nanocages are ideal for the delivery of anticancer drugs due to their lack of immunogenicity and selective interaction with tumor cells (Li et al., 2010;Zhen et al., 2013). However, the current protocols for disassembling and reassembling hHFt to load cargo molecules have still some drawbacks, such as low protein recovery (Zhang et al., 2020;Zhang et al., 2021) . ...
... This drug delivery system was used for simultaneous delivery of doxorubicin (DOX) and mitoxantrone (MTO); which the latter is an anthracycline antibiotic with a broad-spectrum anticancer activity that may intercalate DNA and disrupt topoisomerase II [120]. The system was built in two individual stages: First, DOX was loaded in the interior space of the Apoferritin (AFr) protein with the help of its capability to form a hollow cage upon self-assembly, which dissociates into its constituent parts at a pH of 2.0 and reforms at a pH of 7.4 [121,122], followed by functionalization with folic acid (FA) receptor to give the final TPN particle with a negatively charged surface. Second, the MTO was encapsulated with 97% EE in the cationic solid lipid nanoparticles (cSLN) as a class of SLNs with positive surface charge and the ability to convey various cargos [123,124]. ...
... This drug delivery system was used for simultaneous delivery of doxorubicin (DOX) and mitoxantrone (MTO); which the latter is an anthracycline antibiotic with a broad-spectrum anticancer activity that may intercalate DNA and disrupt topoisomerase II [120]. The system was built in two individual stages: First, DOX was loaded in the interior space of the Apoferritin (AFr) protein with the help of its capability to form a hollow cage upon self-assembly, which dissociates into its constituent parts at a pH of 2.0 and reforms at a pH of 7.4 [121,122], followed by functionalization with folic acid (FA) receptor to give the final TPN particle ...
By utilizing nanoparticles to upload and interact with several pharmaceuticals in varying methods, the primary obstacles associated with loading two or more medications or cargos with different characteristics may be addressed. Therefore, it is feasible to evaluate the benefits provided by co-delivery systems utilizing nanoparticles by investigating the properties and functions of the commonly used structures, such as multi- or simultaneous-stage controlled release, synergic effect, enhanced targetability, and internalization. However, due to the unique surface or core features of each hybrid design, the eventual drug-carrier interactions, release, and penetration processes may vary. Our review article focused on the drug's loading, binding interactions, release, physiochemical, and surface functionalization features, as well as the varying internalization and cytotoxicity of each structure that may aid in the selection of an appropriate design. This was achieved by comparing the actions of uniform-surfaced hybrid particles (such as core-shell particles) to those of anisotropic, asymmetrical hybrid particles (such as Janus, multicompartment, or patchy particles). Information is provided on the use of homogeneous or heterogeneous particles with specified characteristics for the simultaneous delivery of various cargos, possibly enhancing the efficacy of treatment techniques for illnesses such as cancer.
... 4 For example, complexation of the poly(adenosine diphosphateribose) polymerase (PARP) inhibitor olaparib with Cu(II) and subsequent encapsulation performed by the nanoreactor route yielded seven times more drug loaded inside AFt than the encapsulation of the non-complexed organic molecule. 14 This approach was also successful in increasing the stability and the loading efficiency of other anticancer agents such as doxorubicin (DOX) 15 and temozolomide (TMZ) 16 inside horse spleen AFt (HSAFt). Comparison of co-encapsulation using the disassembly-reassembly route with passive diffusion of DOX into pre-formed maghemite-AFt suggested that the latter (nanoreactor) route mostly led to the drug bound at the surface rather than within the cavity, resulting in higher release. ...
Protein capsules are promising drug delivery vehicles for cancer research therapies. Apoferritin (AFt) is a self-assembling 12 nm diameter hollow nanocage with many desirable features for drug delivery, however, control of drug retention inside the protein cage remains challenging. Here we report the encapsulation of copper(ii)-1,10-phenanthroline (Cu(phen)) within the horse spleen AFt (HSAFt) nanocage, by diffusion of the metal through the pores between the protein subunits. Transmission electron microscopy revealed the formation of organised copper adducts inside HSAFt, without affecting protein integrity. These structures proved stable during storage (>4 months at −20 °C). Exposure to physiologically relevant conditions (37 °C) showed some selectivity in cargo release after 24 h at pH 5.5, relevant to the internalisation of AFt within the endosome (60% release), compared to pH 7.4, relevant to the bloodstream (40% release). Co-encapsulation of temozolomide, a prodrug used to treat glioblastoma multiforme, and Cu(phen) enabled entrapment of an average of 339 TMZ molecules per cage. In vitro results from MTT and clonogenic assays identified cytotoxic activity of the Cu(phen), HSAFt–Cu(phen) and HSAFt–Cu(phen)–TMZ adducts against colorectal cancer cells (HCT-116) and glioblastoma cells (U373V, U373M). However, the presence of the metal also contributed to more potent activity toward healthy MRC5 fibroblasts, a result that requires further investigation to assess the clinical viability of this system.
... In mammals, the predominant types of ferritin include HFn and light chain ferritin [21][22][23][24][25]. Among these, HFn has mainly been employed as a nanocarrier for targeted therapeutic and diagnostic applications in cancer [26,27]. Recent studies have demonstrated the successful inhibition of tumor growth in mouse models through the use of drug-loaded HFn [28,29]. ...
Controllable contraception in male animals was demonstrated through the utilization of gold nanorods' photothermal effect to accomplish mild testicular hyperthermia. However, the challenges arising from testicular administration and the non-biodegradability of nanoparticles hinder further clinical implementation. Therefore, a straightforward, non-invasive, and enhanced contraception approach is required. This study explores the utilization of human heavy chain ferritin (HFn) nanocarriers loaded with aggregation-induced emission luminogens (AIEgens) for noninvasive, controllable male contraception guided by Near-Infrared-II (NIR-II) fluorescence imaging. The HFn-caged AIEgens (HFn@BBT) are delivered via intravenous injection and activated by near-infrared irradiation. Lower hyperthermia treatment induces partial damage to the testes and seminiferous tubules, reducing fertility indices by approximately 100% on the 7th day, which gradually recovers to 80% on the 60th day. Conversely, implementation of elevated hyperthermia therapy causes total destruction of both testes and seminiferous tubules, leading to a complete loss of fertility on the 60th day. Additionally, the use of AIEgens in NIR-II imaging offers improved fluorescence efficiency and penetration depth. The findings of this study hold significant promise for the advancement of safe and effective male contraceptive methods, addressing the need for noninvasive and controllable approaches to reproductive health and population control.
... Nanoparticles formulated using proteins [199][200][201], lipids [202][203][204], and polymers [205][206][207] have been commonly used to encapsulate chemotherapeutic drugs. Several nano-formulations including Abraxane ® , Genexol-PM, Caelyx, and Onivyde have been approved for treating metastatic cancer [208]. ...
Simple Summary
The tumor microenvironment plays an important role in drug resistance and supports/promotes tumorigenesis. Stromal modulators in combination with nanomedicine therapeutics have recently been investigated for reprogramming the tumor microenvironment. Here, we review the major stromal components and recent advances in the use of stroma-targeted therapies for cancer treatment.
Abstract
The tumor stroma, or the microenvironment surrounding solid tumors, can significantly impact the effectiveness of cancer therapies. The tumor microenvironment is characterized by high interstitial pressure, a consequence of leaky vasculature, and dense stroma created by excessive deposition of various macromolecules such as collagen, fibronectin, and hyaluronic acid (HA). In addition, non-cancerous cells such as cancer-associated fibroblasts (CAFs) and the extracellular matrix (ECM) itself can promote tumor growth. In recent years, there has been increased interest in combining standard cancer treatments with stromal-targeting strategies or stromal modulators to improve therapeutic outcomes. Furthermore, the use of nanomedicine, which can improve the delivery and retention of drugs in the tumor, has been proposed to target the stroma. This review focuses on how different stromal components contribute to tumor progression and impede chemotherapeutic delivery. Additionally, this review highlights recent advancements in nanomedicine-based stromal modulation and discusses potential future directions for developing more effective stroma-targeted cancer therapies.
... In terms of ferritin purification and production for human consumption, these procedures are very uncommon, and an industrially scalable ferritin purification process may be found only for mammalian recombinant ferritin. In this case, genetically engineered bacteria are being used in order to create recombinant mammalian ferritin for pharmacological usage (Zhen et al., 2013). However, these production procedures are only marginally applicable in the food system. ...
The high prevalence of iron deficiency in humans, alongside the lack of sustainable iron sources call for alternatives. Although understudied, edible insects are high in potentially bioavailable iron. Insect ferritin is thought to make insect-based iron bioavailable (entoferritin). This review examined the use of entoferritin as an iron supplement based on its properties and comparison to mammalian and plant ferritins. Entoferritin is a large, soluble, iron-affine transporting protein complex. These features enable mild entoferritin purification. These purifying methods may not affect other products due to the common structure of edible insect processing chains. The protein complex can accumulate an abundance of bioavailable iron and be absorbed through a human endocytosis mechanism. However, insect ferritin delivery systems into the human iron pool, bioavailability and safety have various possible limits and require further exploration. This paper suggests that entoferritin could help the valorization of edible insects and fight iron deficiency.
... RGD4C targeting peptide-modified ferritin could efficiently target tumors for drug delivery by targeting integrins Rvβ3 and TfR1. 41 Tumor-penetrating peptides can improve the tumor infiltration of chemotherapeutic drugs and enhance anti-tumor efficacy by targeting neuropilin-1, which is overexpressed in tumor cells. 73 Ma et al. 42 modified the penetrating peptide tLyP-1 to the N-terminus of HFn by genetic engineering and loaded paclitaxel (PTX) into HFn nanocages. ...
Background:
Ferritin, a ubiquitously distributed iron storage protein, can specifically target tumor cells through transferrin receptor 1. Due to its rearrangeable nanocage structure, ferritin can be loaded with anticancer drugs. Combined with amino acid modifications on the outer- and/or inner-spaces of the nanocage, ferritins can be further coupled with antigens, antibodies, and nucleotide sequences. Since ferritin is naturally presented in the human body, when used in vivo, ferritin exhibits good biocompatibility, and no immunogenic response occurs. These makes ferritin an ideal nanocarrier which shows broad application prospects in cancer therapy.
Methods:
In this study, to find articles, a search was made in PubMed with the keywords ferritin, drug delivery, drug delivery, and cancer treatment.
Results:
According to the investigation, some studies suggest that ferritin can be loaded with drugs and targeted for delivery to tumor tissue. Therefore, ferritin nanocarriers loaded with drugs can be used in chemotherapy, photodynamic therapy (PDT), photothermal therapy (PTT) and immunotherapy. Importantly, the specific targeting of ferritin nanocarriers to tumor cells increases the effectiveness of related therapies and reduces side effects.
Conclusions:
We conclude in this paper that the superior properties of ferritin nanocarriers as an emerging drug delivery system make them a promising cancer treatment strategy. In the future, it is worth conducting clinical trials to further investigate the safety and efficacy of ferritin nanocarriers in patients.
... Metal-assisted passive loading method was also proven useful for non-metal molecules' encapsulation. Dox was pre-complexed with a transition metal Cu(II) to generate Cu(II)-Dox complex, which traversed ferritin threefold hydrophilic channels by passive diffusing and was encapsulated with a quite high loading rate of 73%, while pre-incubation of ferritin with Cu(II) alone significantly reduced Cu(II)-Dox loading rate to 8.28%, suggesting the encapsulating efficiency of Cu(II)-Dox was highly depend on metal-binding sites on the inner surface which could be competitively occupied by Cu(II) [50]. Two other molecules, navitoclax [39] and ATP [52], were also reported to be encapsulated by metal ion's assisting. ...
Ferritin is an endogenous protein which is self-assembled by 24 subunits into a highly uniform nanocage structure. Due to the drug-encapsulating ability in the hollow inner cavity and abundant modification sites on the outer surface, ferritin nanocage has been demonstrated great potential to become a multi-functional nanomedicine platform. Its good biocompatibility, low toxicity and immunogenicity, intrinsic tumor-targeting ability, high stability, low cost and massive production, together make ferritin nanocage stand out from other nanocarriers. In this review, we summarized ferritin-based nanomedicine in field of disease diagnosis, treatment and prevention. The different types of drugs to be loaded in ferritin, as well as drug-loading methods were classified. The strategies for site-specific and non-specific functional modification of ferritin were investigated, then the application of ferritin for disease imaging, drug delivery and vaccine development were discussed. Finally, the challenges restricting the clinical translation of ferritin-based nanomedicines were analyzed.
... 39,40 Ferritin coated with RGDs can also effectively target tumor tissues via the interaction between RGDs and integrins. 41,42 Furthermore, attempts have been made to enhance NP deposition, cellular uptake, and intracellular drug release by thermal, electric, magnetic, optical, and acoustic activation. Studies have shown that the deposition of ferrimagnetic NPs in tumor tissues can improve the treatment efficiency of microwave ablation. ...
Background:
A good understanding of the adhesion behaviors of the nanocarriers in microvessels in chemo-hyperthermia synergistic therapy is conducive to nanocarrier design for targeted drug delivery.
Methods:
In this study, we constructed an artificial blood vessel system using gelatins with a complete endothelial monolayer formed on the inner vessel wall. The numbers of adhered NPs under different conditions were measured, as well as the interaction forces between the arginine-glycine-aspartic acid (RGD) ligands and endothelial cells.
Results:
The experimental results on the adhesion of ligand-coated nanoparticles (NPs) with different sizes and morphologies in the blood vessel verified that the gelatin-based artificial vessel possessed good cytocompatibility and mechanical properties, which are suitable for the investigation on NP adhesion characteristics in microvessels. When the temperature deviated from 37 °C, an increase or decrease in temperature resulted in a decrease in the number of adhered NPs, but the margination probability of NP adhesion increased at high temperatures due to the enhanced Brownian movement and flow disturbance. It is found that the effect of cooling was less than that of heating according to the observed changes in cell morphology and a decrease in cell activity under the static and perfusion culture conditions within the temperature range of 25 °C-43 °C. Furthermore, the measurement results of change in the RGD ligand-cell interaction with temperature showed good agreement with those in the number of adhered NPs.
Conclusion:
The Findings suggest that designing ligands that can bind to the receptor and are least susceptible to temperature variation can be an effective means to enhance drug retention.
... Homopolymeric Fts constituted by human H chains (hHFt) are considered very promising nanosystems for drug delivery (Li et al., 2010). The advantages of using these proteins as drug carriers are related to a series of features (Zhen et al., 2013). hHFt is biocompatible: it is already present in the human body; thus, it is recognized by the immune system, and its administration can avoid undesired reactions and side effects. ...
For their easy and high-yield recombinant production, their high stability in a wide range of physico-chemical conditions and their characteristic hollow structure, ferritins (Fts) are considered useful scaffolds to encapsulate bioactive molecules. Notably, for the absence of immunogenicity and the selective interaction with tumor cells, the nanocages constituted by the heavy chain of the human variant of ferritin (hHFt) are optimal candidates for the delivery of anti-cancer drugs. hHFt nanocages can be disassembled and reassembled in vitro to allow the loading of cargo molecules, however the currently available protocols present some relevant drawbacks. Indeed, protein disassembly is achieved by exposure to extreme pH (either acidic or alkaline), followed by incubation at neutral pH to allow reassembly, but the final protein recovery and homogeneity are not satisfactory. Moreover, the exposure to extreme pH may affect the structure of the molecule to be loaded. In this paper, we report an alternative, efficient and reproducible procedure to reversibly disassemble hHFt under mild pH conditions. We demonstrate that a small amount of sodium dodecyl sulfate (SDS) is sufficient to disassemble the nanocage, which quantitatively reassembles upon SDS removal. Electron microscopy and X-ray crystallography show that the reassembled protein is identical to the untreated one. The newly developed procedure was used to encapsulate two small molecules. When compared to the existing disassembly/reassembly procedures, our approach can be applied in a wide range of pH values and temperatures, is compatible with a larger number of cargos and allows a higher protein recovery.
... RGD is frequently used for the modification of NPs to target different kinds of tumors. High affinity and specificity attachment of RGD to integrin receptors, which are highly overexpressed on the various types of tumor endothelium cells, enables this peptide to be employed as targeting moieties for tumor nanomedicine treatment [185][186][187]. Related to this, in a study, RGD conjugated lipid-polymer, hybrid nanocarrier represented as a good target agent for integrin-expressing HCC cells [188]. ...
Human hepatocellular carcinoma (HCC) has been recognized as one of the significant causes of mortality around the globe. In recent years, extensive research has been carried out to find a deep-rooted perception of mechanisms involved in the pathogenesis of the disease to develop novel strategies for compassionate diagnostic and therapeutic tools. In this regard, nanotechnology has presented great opportunities in HCC diagnosis and treatment by selective and specific targeting, and efficient delivery to reach sufficient doses in targeted tumor areas without adverse influences or minimal damage to normal cells. Herein, different Nanomedicine-mediated strategies for HCC therapy are reviewed including stem cell therapy, photodynamic and photothermal therapy, sonodynamic therapy, gene therapy, and theranostics. Also, recent developments in nanomedicine are discussed for the preparation of HCC diagnostic and drug delivery systems, and their clinical status and future perspective are highlighted.
... Besides their physiological function, the nanocage properties of ferritins have been investigated in several different biotechnological applications, such as drug delivery vectors (2,16), scaffolds for vaccine development (17) and tools for bioimaging (18,19). Concerning these tools, many studies have been carried out on quantum dots, gold nanoparticles and luminescent metal chelators (20,21), whereas only a few studies have been done on ferritinbased constructs to use as smart luminescent probes. ...
Ferritin nanoparticles play many important roles in theranostic and bioengineering applications and have been successfully used as nanovectors for the targeted delivery of drugs due to their ability to specifically bind the transferrin receptor (TfR1, or CD71). They can be either genetically or chemically modified for encapsulating therapeutics or probes in their inner cavity. Here, we analyzed a new engineered ferritin nanoparticle, made of the H chain mouse ferritin (HFt) fused with a specific lanthanide binding tag (LBT). The HFt‐LBT has one high affinity lanthanide binding site per each of the 24 subunits and a tryptophane residue within the tag that acts as an antenna able to transfer the energy to the lanthanide ions via a LRET process. In this study, among lanthanides, we selected europium for its red emission that allows to reduce overlap with tissue auto‐fluorescence. Steady state emission measurements and time‐resolved emission spectroscopy have been employed to investigate the interaction between the HFt‐LBT and the Eu3+ ions. This allowed us to identify the Eu3+ energy states involved in the process and to pave the way for the future use of HFt‐LBT Eu3+ complex in theranostics.
... Besides, modifying tumor cell targeting moieties on the NP surface is a widely recognized method to improve the specificity and effectiveness of the NP uptake [41]. For example, the RGD (Arg-Gly-Asp) peptide has excellent active targeting ability due to its specific binding to αvβ3 integrin, which is highly expressed on tumor cells and tumor neovascular endothelial cells [39,[41][42][43]. Therefore, developing "all-in-one" nanoplatform with tumor-specificity and high-targeting ability for CDT enhanced phototheranostics is of great significance to improve the theranostic efficiency. ...
Tumor phototheranostics holds a great promise on account of its high spatiotemporal resolution, tumor-specificity, and noninvasiveness. However, physical limitation of light penetration and “always on” properties of conventional photothermal-conversion agents usually cause difficulty in accurate diagnosis and completely elimination of tumor. Meanwhile, nanozymes mediated Fenton reactions can well utilize the tumor microenvironment (TME) to generate hydroxyl radicals for chemodynamic therapy (CDT), but limited by the concentration of H2O2 in TME and the delivery efficiency of nanozymes. To overcome these problems, a dual-targeting nanozyme (FTRNPs) is developed for tumor-specific in situ theranostics, based upon the assembling of ultrasmall Fe3O4 nanoparticles, 3,3’,5,5’-tetrameth-ylbenzidine (TMB) and the RGD peptide. The FTRNPs after H2O2 treatment exhibits superior photothermal stability and high photothermal conversion efficiency (η = 50.9%). FTRNPs shows extraordinary accumulation and retention in the tumor site by biological/physical dual-targeting, which is 3.54-fold higher than that without active targeting. Cascade-dual-response to TME for nanozymes mediated Fenton reactions and TMB oxidation further improves the accuracy of both photoacoustic imaging and photothermal therapy (PTT). The tumor inhibition rate of photo-chemodynamic therapy is ~ 97.76%, which is ~ 4-fold higher than that of PTT or CDT only. Thus, the combination of CDT and PTT to construct “turn on” nanoplatform is of great significance to overcome their respective limitations. Considering its optimized “all-in-one” performance, this new nanoplatform is expected to provide an advanced theranostic strategy for the future treatment of cancers.
Graphical abstract
... Très bien connue et caractérisée, c'est une structure de choix pour de nombreuses applications, notamment en nanomédecine 37 . Zhen et al. 38 ont, par exemple, recouru à la ferritine comme système de délivrance pour le traitement du cancer en encapsulant la doxorubicine, une molécule anticancéreuse. Kanekiyo et al. 39 ont quant à eux développé un vaccin qui peut induire la production d'anticorps anti-H1N1 basé sur une fusion de la ferritine avec l'hémagglutinine du virus, une protéine de surface. ...
Les organismes vivants ont développé au cours de l’évolution une très large gamme de protéines capables de s’autoassembler et de former des objets 3D tels que les capsides de virus. Dans ces travaux, nous avons principalement produit deux autoassemblages en système d’expression bactérien et envisagé deux projets distincts. Un premier projet s’intéresse à un assemblage appelé R-body issu de bactéries. Le R-body est une structure protéique qui forme un ruban enroulé de 500 nm à pH neutre, capable de s’étendre en tube de20 µm de long dans un milieu plus acide. In vivo, cette propriété lui permet de perforer des membranes biologiques. Nous avons ainsi investigué la possibilité d’exploiter des R-bodies pour créer une vésicule de délivrance contrôlée par la lumière. Le second projet de cette thèse concerne le développement de particules pseudovirales (VLPs) pour diriger le comportement cellulaire. La VLP du bactériophage AP205 utilisée permet de fusionner des peptides bioactifs à l’extrémité des protéines d’enveloppe qui seront ensuite présentés à l’extérieur de la particule. Par clonage moléculaire, nous avons ajouté aux particules des peptides bioactifs connus pour induire une réponse similaire à leur protéine d’origine (adhésion cellulaire, différenciation) et nous avons cherché à mettre en avant l’efficacité d’une telle approche sur la reconnaissance et le comportement des cellules. Les résultats obtenus montrent que les VLPs peuvent être utilisées pour contrôler l’adhésion cellulaire et ouvrent de nombreuses perspectives sur l’utilisation de la VLP AP205 comme une plateforme modulaire pour contrôler le comportement des cellules à la surface de biomatériaux.
... Finally, the medicine was released and played its role in disease. For example, Zhen et al. (2013) used ferritin to encapsulate doxorubicin as a drug delivery vehicle. Engineered ferritin nanoparticles for the bioluminescence tracking of nanomedicine delivery in cancer (Bellini et al., 2020). ...
Triggering receptor expressed on myeloid cells-1 (TREM-1) regulates inflammation and promotes a vigorous immune response. GF9 is one of the peptides that inhibit the mTREM-1 signaling pathway, thus reducing the inflammatory mediators in diseases including sepsis. Nanotechnology could offer a new complementary strategy for diseases. Streptomycin is also one treatment of sepsis. However, the role of nanoparticles delivered GF9 combined with streptomycin on sepsis had never been discovered. In the present study, cecal ligation and puncture (CLP) and lipopolysaccharide [LPS, Escherichia coli (E. coli) O111:B4] sepsis models were constructed. SDS-PAGE was used to evaluate the size of nano drugs; Western blot was used to detect the protein levels of MMP2 and TREM-1 in cells. The levels of TNF-α and IL-6 were detected by ELISA. Histopathological changes were observed by HE staining. And the nanomedicines of GF9-HFn/Str were successfully constructed. The size of GF9-HFn/Str is 40 kD. The ferritin-based nanoparticle plays a vital role in delivering streptomycin into cells and tissues. GF9 (1.6 μM) and streptomycin (40 μM) co-delivery nanomedicine showed a better effect on promoting overall survival, decreasing E. coli, significantly suppressed the expression levels of inflammatory factors (TNF-α and IL-6), and can reduce lung injury. Our study demonstrated that combination delivery of nanomedicine GF9 and streptomycin have a better effect on overall survival rate, anti-inflammatory, and anti-bacterial in sepsis. Our present study revealed a new potential therapeutic method for sepsis.
... The existence of a temperature-triggerable channel in mammalian H-subunit FRT allows entry of larger drug molecules (doxorubicin, epirubicin, cisplatin or oxaliplatin) that can withstand the increased temperature (60 • C for 4 h for HsaH) [13]. A successful approach has also utilized a pre-complexation of the drug molecules (doxorubicin [38] and everolimus [39]) with Cu (II) , facilitating passive loading through the charged pores. An increased hydrostatic pressure has also been used to load doxorubicin into HsaH [40]. ...
Understanding the reversible reassembly properties of ferritins (FRTs) is crucial for enabling their applicability in nanomedicine. These properties include drug loading capabilities and also subsequent payload release in desired site-of-action. Thus, the presented study is focused on understanding the disassembly of recombinantly produced FRTs of varying origin and subunit composition, i.e. equine FRT composed of 22 Land 2 H-subunits (EcaLH) or 24 L-subunits (EcaL), human FRT composed of 24 H-subunits (HsaH) and archaeal Pyrococcus furiosus FRT (Pfu). Disassembly was distinctly influenced by pH and ionic strength. Except for Pfu, the disassembly kinetics in acidic pH is rapid upon reaching an innate threshold, reaching the final state within minutes. Disassembly kinetics in basic pH is slower. Pfu is partially disassembled within the entire pH range. While equine FRT disassembles into free subunits, HsaH disassembles into subunit clusters. EcaL and Pfu form large aggregates during disassembly in mildly acidic pH, although basic pH causes partial disassembly without aggregation, suggesting usability for basic pH-triggered drug loading. We show that in human serum/plasma, FRTs readily form protein corona, hampering their uptake. Interestingly, we found out that archaeal Pfu likely exhibits similar receptor affinity as mammalian FRTs. Further, in vivo toxicity and biodistribution study of a single dose of FRTs demonstrated very low toxicity of FRTs and their preferential liver/kidney bioaccumulation.
... [125] Dox Copper-Dox complexes capable to bind to apoferritin binding sites. [126] E2 subunit pyruvate dehydrogenase Dox (6-Maleinimidocaproyl) hydrazone derivative of Dox covalently coupled to the interior cavity of the cage. ...
Proteins and peptides are attractive chemical building blocks to encapsulate and protect active substances thanks to their biocompatibility, biodegradability, low immunogenicity, and added functionality compared to synthetic polymers. This review provides a comprehensive overview of micro- and nanocapsules predominantly made of proteins—both natural and artificially produced—and peptides, detailing their different fabrication techniques and possible applications in various fields, including food technology and healthcare. Emphasis is given on the capability of proteins and peptides to assemble into capsular structures in the absence (e.g., protein cages and polypeptide-based coacervates) or presence of a template, as well as on the physical nature of the carriers core, i.e., gaseous, liquid, or solid.
... The naturally occurring selfassembling iron-storage protein, ferritin nanocage is of particular importance because of its high thermal and chemical stability as well as its ability to encapsulate various organic and inorganic compounds, which have wide applications in biotechnology and other areas. [15,16] For example, the anticancer drug doxorubicin encapsulated inside the ferritin nanocage was shown to be more effective than free doxorubicin for anticancer activity, [17] magnetoferritin was shown to act as a contrast agent for magnetic resonance imaging, [18] Pd nanoclusters embedded inside the ferritin nanocage was shown to be active towards size-selective olefin hydrogenation. [7] Structural and mechanistic studies of incorporation of various guest molecules including metal complexes into the core of the apo-ferritin nanocage have been reported previously. ...
The effect of the mutation at the core of the ferritin nanocage (apo‐rHLFr) on the uptake of IrCp* has been investigated by structural and spectroscopic methods. Site‐specific mutations of two polar residues viz., Asp38 and Arg52 were investigated. The uptake of IrCp* was increased by about 1.5‐fold on mutation of Arg52 by His compared to the wild‐type variant, while mutation of Asp38 by His had no effect on the uptake. All the variants of the Ir‐embedded ferritin cages remained as stable as the wild‐type analogue. These hybrid bio‐nanocages of ferritin were found to efficiently catalyze transfer hydrogenation of various substituted acetophenones forming the corresponding chiral alcohols with up to 88 % conversion and 70 % enantioselectivity. An electron‐withdrawing substituent on the reactant enhanced the Turnover frequency of the reaction. Molecular docking analyses suggested that the substrate binds in different orientations at the active site in different mutants of the nanocage.
Controllable protein nanoarchitectonics refers to the process of manipulating and controlling the assembly of proteins at the nanoscale to achieve domain‐limited and accurate spatial arrangement. In nature, many proteins undergo precise self‐assembly with other structural domains to engage in synergistic physiological activities. Protein nanomaterials prepared through protein nanosizing have received considerable attention due to their excellent biocompatibility, low toxicity, modifiability, and versatility. This review focuses on the fundamental strategies used for controllable protein nanoarchitectinics, which include computational design, self‐assembly induction, template introduction, complexation induction, chemical modification, and in vivo assembly. Precise controlling of the nanosizing process has enabled the creation of protein nanostructures with different dimensions, including 0D spherical oligomers, 1D nanowires, nanorings, and nanotubes, as well as 2D nanofilms, and 3D protein nanocages. The unique biological properties of proteins hold promise for diverse applications of these protein nanomaterials, including in biomedicine, the food industry, agriculture, biosensing, environmental protection, biocatalysis, and artificial light harvesting. Protein nanosizing is a powerful tool for developing biomaterials with advanced structures and functions.
Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.
Simultaneous detection of live and dead bacteria is a huge challenge for food safety. To solve this issue, an all-in-one biosensor for bacteria was developed using the phage-apoferritin@CuO2 (phage-Apo@CP) probe on an antimicrobial peptide (AMP)/MXenes-modified detection platform. With the specific recognition of AMP and phage-Apo@CP, the biosensor for the target Escherichia coli O157:H7 (E. coli O157:H7) presented multi-mode (bioluminescent, colorimetric, and electrochemical) signals to simultaneously measure live and dead bacteria. The bioluminescent signal caused by the adenosine triphosphate (ATP) from the bacteria was used to quantify live bacteria. The colorimetric and voltammetric signals triggered by ·OH and Cu2+ from the probe with the assistance of acid could rapidly screen and quantitative determination of total E. coli O157:H7 concentration. Thus, the dead one was obtained according to the total and live ones. All three signals could be mutually corrected to improve the accuracy. The biosensor was successfully used for on-site measurement of live and dead E. coli O157:H7 in food samples with the limit of detection of 30 CFU/mL for live ones and 6 CFU/mL for total bacteria within 50 min. This work presents a novel pathway for rapid and simultaneous quantification of both live and dead bacteria.
Since its discovery more than 85 years ago, ferritin has principally been known as an iron storage protein. However, new roles, beyond iron storage, are being uncovered. Novel processes involving ferritin such as ferritinophagy and ferroptosis and as a cellular iron delivery protein not only expand our thinking on the range of contributions of this protein but present an opportunity to target these pathways in cancers. The key question we focus on within this review is whether ferritin modulation represents a useful approach for treating cancers. We discussed novel functions and processes of this protein in cancers. We are not limiting this review to cell intrinsic modulation of ferritin in cancers, but also focus on its utility in the trojan horse approach in cancer therapeutics. The novel functions of ferritin as discussed herein realize the multiple roles of ferritin in cell biology that can be probed for therapeutic opportunities and further research.
The current advancement in diagnostic research by implementing nanotechnology encourages researchers worldwide to focus towards new possibilities of utilizing nano formulation for advanced imaging and diagnosis for cancer. This concept initiates the development of multimodal theranostic nanoparticles which has both therapy and diagnosis property in cancer treatment. The potential advantage of these theranostic nanoparticles over other nanoformulations they are highly efficient transporters and cargos both imaging and therapeutic agents as encapsulated and the intrinsic molecular property aiding effective diagnosis. This dual functional capacity of theranostic nano particles accounts their usage as personalized medicine. In this book chapter we tried to included details of various bio inspired theranostic nano particles and their application in cancer therapy. The contents covered are Bio-inspired nanoparticles used in cancer (Liposome, Lipid based theranostic nanoparticles (LNPs), Solid form lipid nanoparticles (SLNs), Lipid based Nano capsules (LNCs), Lipid-nano structure (NLCs), Lipid micelles), Protein based theranostic nanoparticles, Viral nanoparticles (VNPs), Oligonucleotide theranostic nanoparticles, Peptide theranostic nanoparticles. Whereas In-organic theranostic nanoparticles including Gold theranostic nanoparticles (AuNPs), Silver theranostic nanoparticles (AgNPs), Iron oxide nano particlesKeywordsNanoparticlesTheranosticInorganicOrganic-nanoparticlesCancer
The biocompatible protein nanocarrier with homogeneous particle size is a promising candidate material for the delivery of targeted drugs to tumors. Doxorubicin (DOX) is a commonly prescribed anthracycline antitumor drug, although it may cause nephrotoxicity and cardiotoxicity. The Chinese herbal remedy ursolic acid (UA), a pentacyclic triterpenoid with anticancer action, has been used as a potential drug sensitizer to increase the effectiveness of chemotherapy and pharmacological therapy. Therefore, the dose of DOX can be reduced by compatibility with UA to lower its side effects. Ferritin binds to tumor cells through an interaction with the transferrin receptor 1 (TfR1), which is overexpressed in human cancer cells. In this study, the hydrophobic drug UA and the hydrophilic drug DOX were successfully encapsulated into the ferritin inner cavity using the thermal treatment method incubated at 60 °C for 4 h. The results demonstrated that loaded ferritin could specifically enter breast cancer cells MCF-7 and non-small-cell lung cancer cells A549 in comparison with free UA and DOX, enhancing their therapeutic effects. The loading ratio of two drugs was optimized in the constructed nanocarriers, and the effectiveness of the constructed nanodrugs in inhibiting tumor proliferation was verified by cell apoptosis and three-dimensional (3D) tumor spheroids studies. For the first time, the hydrophilic and hydrophobic drugs were loaded simultaneously within unmodified ferritin without other addition of additives, which would reduce the toxic side effects of DOX and enhance its therapeutic effect. This study also showed that the ferritin-based nanocarrier has potential for drug delivery to tumors.
Proteins and enzymes are versatile biomaterials for a wide range of medical applications due to their high specificity for receptors and substrates, high degradability, low toxicity, and overall good biocompatibility. Protein nanoparticles are formed by the arrangement of several native or modified proteins into nanometer‐sized assemblies. In this review, we will focus on artificial nanoparticle systems, where proteins are the main structural element and not just an encapsulated payload. While under natural conditions, only certain proteins form defined aggregates and nanoparticles, chemical modifications or a change in the physical environment can further extend the pool of available building blocks. This allows the assembly of many globular proteins and even enzymes. These advances in preparation methods led to the emergence of new generations of nanosystems that extend beyond transport vehicles to diverse applications, from multifunctional drug delivery to imaging, nanocatalysis and protein therapy.
Due to its beneficial pharmacological properties, ferritin (Ftn) is considered as an interesting drug delivery vehicle to alleviate the cardiotoxicity of doxorubicin (DOX) in chemotherapy. However, the encapsulation of DOX in Ftn suffers from heavy precipitation and low protein recovery yield which limits its full potential. Here, a new DOX encapsulation strategy by cysteine-maleimide conjugation is proposed. In order to demonstrate that this strategy is more efficient compared to the other approaches, DOX is encapsulated in Ftn variants carrying different surface charges. Furthermore, in contrast to the common belief, this data show that DOX molecules are also found to bind non-specifically to the surface of Ftn. This can be circumvented by the use of Tris(2-carboxyethyl)phosphine (TCEP) during encapsulation or by washing with acidic buffer. The biocompatibility studies of the resulting DOX Ftn variants in MCF-7 and MHS cancer cells shows a complex relationship between the cytotoxicity, the DOX loading and the different surface charges of Ftn. Further investigation on the cell uptake mechanism provides reasonable explanations for the cytotoxicity results and reveals that surface charging of Ftn hinders its transferrin receptor 1 (TfR-1) mediated cellular uptake in MCF-7 cells.
Nanoparticles are used as carriers for the delivery of drugs and imaging agents. Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents and other cargos. However, recombinant ferritin currently costs ~3.5 million € g⁻¹, presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain (FTH1) by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5′-UTRs in plant cell packs, and selected the best-performing construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by a ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg⁻¹ fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~ 3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for therapeutic and diagnostic agents, which is suitable for further stability and functionality testing in vitro and in vivo.
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Inefficient tumor-targeted delivery and uncontrolled drug release are the major obstacles in cancer chemotherapy. Herein, inspired by the targeting advantage of coronavirus from its size and coronal structure, a coronal biological metal-organic framework nanovehicle (named as corona-BioMOF) is constructed for improving its precise cancer targeting ability. The designed corona-BioMOF is constructed as the carriers-encapsulated carrier model by inner coated with abundant protein-nanocaged doxorubicin particles and external decorated with high-affinity apoferritin proteins to form the spiky surface for constructing the specific coronal structure. The corona-BioMOF shows a higher affinity and enhanced targeting ability towards receptor-positive cancer cells compared to that of MOF-drug composites without spiky surface. It also exhibits the hierarchical wrapping pattern-endowed controlled lysosome-specific drug release and remarkable tumor lethality in vivo. Moreover, water-induced surface defect-based protein handle mechanism is first proposed to shape the coronal-BioMOF. This work will provide better inspiration for nanovehicle construction and be broadly useful for clinical precision nanomedicine.
Protein assemblies have drawn much attention as platforms for biomedical applications, including gene/drug delivery and vaccine, due to biocompatibility and functional diversity. Here, the construction and functionalization of a protein assembly composed of human clathrin heavy chain and light chain for a targeted protein delivery, is presented. The clathrin heavy and light chains are redesigned and associated with each other, and the resulting triskelion unit further self‐assembled into a clathrin assembly with the size of about 28 nm in diameter. The clathrin assembly is dual‐functionalized with a protein cargo and a targeting moiety using two different orthogonal protein–ligand pairs through one‐pot reaction. The functionalized clathrin assembly exhibits about a 900‐fold decreased KD value for a cell‐surface target due to avidity compared to a native targeting moiety. The utility of the clathrin assembly is demonstrated by an efficient delivery of a protein cargo into tumor cells in a target‐specific manner, resulting in a strong cytotoxic effect. The present approach can be used in the creation of protein assemblies with multimodality. A new type of protein self‐assembly using human clathrin is developed. Using two different orthogonal protein–ligand pairs, both heavy and light chains of a clathrin triskelion are redesigned for a dual‐functionalization with functional biomolecules. The resulting dual‐functionalized clathrin assembly shows a target‐specific protein delivery and exemplifies its utility as a protein delivery platform.
Durable glioblastoma multiforme (GBM) management requires long-term chemotherapy after surgery to eliminate remaining cancerous tissues. Among chemotherapeutics, temozolomide is considered as the first-line drug for GBM therapy, but the treatment outcome is not satisfactory. Notably, regorafenib, an oral multi-kinase inhibitor, has been reported to exert a markedly superior effect on GBM suppression compared with temozolomide. However, poor site-specific delivery and bioavailability significantly restrict the efficient permeability of regorafenib to brain lesions and compromise its treatment efficacy. Therefore, human H-ferritin (HFn), regorafenib, and Cu2+ are rationally designed as a brain-targeted nanoplatform (HFn-Cu-REGO NPs), fulfilling the task of site-specific delivery and manipulating autophagy and cuproptosis against GBM. Herein, HFn affords a preferential accumulation capacity to GBM due to transferrin receptor 1 (TfR1)-mediated active targeting and pH-responsive delivery behavior. Moreover, regorafenib can inhibit autophagosome-lysosome fusion, resulting in lethal autophagy arrest in GBM cells. Furthermore, Cu2+ not only facilitates the encapsulation of regorafenib to HFn through coordination interaction but also disturbs copper homeostasis for triggering cuproptosis, resulting in a synergistical effect with regorafenib-mediated lethal autophagy arrest against GBM. Therefore, this work may broaden the clinical application scope of Cu2+ and regorafenib in GBM treatment via modulating autophagy and cuproptosis.
Fundamental clinical areas such as drug delivery and regenerative medicine require biocompatible materials as mechanically stable scaffolds or as nanoscale drug carriers. Among the wide set of emerging biomaterials, polypeptides offer enticing properties over alternative polymers, including full biocompatibility, biodegradability, precise interactivity, structural stability and conformational and functional versatility, all of them tunable by conventional protein engineering. However, proteins from non-human sources elicit immunotoxicities that might bottleneck further development and narrow their clinical applicability. In this context, selecting human proteins or developing humanized protein versions as building blocks is a strict demand to design non-immunogenic protein materials. We review here the expanding catalogue of human or humanized proteins tailored to execute different levels of scaffolding functions and how they can be engineered as self-assembling materials in form of oligomers, polymers or complex networks. In particular, we emphasize those that are under clinical development, revising their fields of applicability and how they have been adapted to offer, apart from mere mechanical support, highly refined functions and precise molecular interactions.
Background
Naturally occurring protein cages, both viral and non-viral assemblies, have been developed for various pharmaceutical applications. Protein cages are ideal platforms as they are compatible, biodegradable, bioavailable, and amenable to chemical and genetic modification to impart new functionalities for selective targeting or tracking of proteins. The ferritin/apoferritin protein cage, plant-derived viral capsids, the small Heat shock protein, albumin, soy and whey protein, collagen, and gelatin have all been exploited and characterized as drug-delivery vehicles. Protein cages come in many shapes and types with unique features such as unmatched uniformity, size, and conjugations.
Objectives
The recent strategic development of drug delivery will be covered in this review, emphasizing polymer-based, specifically protein-based, drug delivery nanomedicine platforms. The potential and drawbacks of each kind of protein-based drug-delivery system will also be highlighted.
Methods
Research examining the usability of nanomaterials in the pharmaceutical and medical sectors were identified by employing bibliographic databases and web search engines.
Results
Rings, tubes, and cages are unique protein structures that occur in the biological environment and might serve as building blocks for nanomachines. Furthermore, numerous virions can undergo reversible structural conformational changes that open or close gated pores, allowing customizable accessibility to their core and ideal delivery vehicles.
Conclusion
Protein cages' biocompatibility and their ability to be precisely engineered indicate they have significant potential in drug delivery and intracellular administration.
Cancer is a disease that seriously threatens human health. Based on the improvement of traditional treatment methods and the development of new treatment modes, the pattern of cancer treatment is constantly being optimized. Nanomedicine plays an important role in these evolving tumor treatment modalities. In this article, we outline the applications of nanomedicine in three important tumor-related fields: chemotherapy, gene therapy, and immunotherapy. According to the current common problems, such as poor targeting of first-line chemotherapy drugs, easy destruction of nucleic acid drugs, and common immune-related adverse events in immunotherapy, we discuss how nanomedicine can be combined with these treatment modalities, provide typical examples, and summarize the advantages brought by the application of nanomedicine.
Nanoscale technologies are crucial for the characterization and fabrication of biomaterials that are useful in targeted drug delivery systems. New materials enable the delivery of therapeutic agents to specific tissues and cells in order to treat a range of diseases.
Bionanotechnology: Next-Generation Therapeutic Tools provides a quick overview of the use of nanomaterials in modern drug delivery and targeted drug therapy systems. The book starts with an overview of nanomaterial toxicity with subsequent chapters detailing their applications in nanomedicine. Concepts such as immunotherapy, cancer theranostics, molecular imaging, aptamers and viral nanoparticles are highlighted in specific chapters. The simplified presentation along with scientific references makes this book ideal for pharmacology and biomedical engineering scholars and life science readers.
Advanced treatments based on immune system manipulation, gene transcription and regulation, specific organ and cell targeting, and/or photon energy conversion have emerged as promising therapeutic strategies against a range of challenging diseases. Naturally derived macromolecules (e.g., proteins, lipids, polysaccharides, and polyphenols) have increasingly found use as fundamental building blocks for nanostructured particles as their advantageous properties, including biocompatibility, biodegradability, inherent bioactivity, and diverse chemical properties make them suitable for advanced therapeutic applications. This review provides a timely and comprehensive summary of the use of a broad range of natural building blocks in the rapidly developing field of advanced therapeutics with insights specific to nanostructured particles. We focus on an up-to-date overview of the assembly of nanostructured particles using natural building blocks and summarize their key scientific and preclinical milestones for advanced therapies, including adoptive cell therapy, immunotherapy, gene therapy, active targeted drug delivery, photoacoustic therapy and imaging, photothermal therapy, and combinational therapy. A cross-comparison of the advantages and disadvantages of different natural building blocks are highlighted to elucidate the key design principles for such bio-derived nanoparticles toward improving their performance and adoption. Current challenges and future research directions are also discussed, which will accelerate our understanding of designing, engineering, and applying nanostructured particles for advanced therapies.
Traditional anticancer treatments have several limitations, but cancer is still one of the deadliest diseases. As a result, new anticancer drugs are required for the treatment of cancer. The use of metal nanoparticles (NPs) as alternative chemotherapeutic drugs is on the rise in cancer research. Metal NPs have the potential for use in a wide range of applications. Natural or surface-induced anticancer effects can be found in metals. The focus of this review is on the therapeutic potential of metal-based NPs. The potential of various types of metal NPs for tumor targeting will be discussed for cancer treatment. The in vivo application of metal NPs for solid tumors will be reviewed. Risk factors involved in the clinical application of metal NPs will also be summarized.
The effect of the mutation at the core of the ferritin nanocage (apo-rHLFr) on the uptake of IrCp* has been investigated by structural and spectroscopic methods. Site-specific mutations of two polar residues viz., Asp38 and Arg52 were investigated. The uptake of IrCp* was increased by about 1.5-fold on mutation of Arg52 by His compared to the wild-type variant, while mutation of Asp38 by His had no effect on the uptake. All the variants of the Ir-embedded ferritin cages remained as stable as the wild type analogue. These hybrid bio-nanocages of ferritin were found to efficiently catalyze transfer hydrogenation of various substituted acetophenones forming corresponding chiral alcohols with up to 88% conversion and 70% enantioselectivity. Electron-withdrawing substituent on the reactant enhanced the TOF of the reaction. Molecular docking analyses suggested that the substrate binds in different orientations at the active site in different mutants of the nanocage.
Methods that leverage bone marrow mesenchymal stem cells (BMSCs) and stimulating factor kartogenin (KGN) for chondrocyte differentiation have paved the way for cartilage repair. However, the scarce carriers for efficiently bridging the two components significantly impede their further application. Therefore, one kind of bifunctional ferritin has designed and synthesized: RC‐Fn, a genetically engineered ferritin nanocage with RGD peptide and WYRGRL peptide on the surface. The RGD can target the integrin αvβ3 of BMSCs and promote proliferation, and the WYRGRL peptide has an inherent affinity for the cartilage matrix component of collagen II protein. RC‐Fn nanocages have an ideal size for penetrating the proteoglycan network of cartilage. Thus, intra‐articularly injected RC‐Fn with KGN loading can convert the articular cavity from a barrier into a reservoir to prevent rapid release and clearance of KGN and exogenous BMSCs, which results in efficient and persistent chondrogenesis in cartilage regeneration.
Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated.
Engineered nanoparticles have been used to provide diagnostic,
therapeutic and prognostic information about the status of disease.
Nanoparticles developed for these purposes are typically modified with
targeting ligands (such as antibodies, peptides or small molecules) or
contrast agents using complicated processes and expensive reagents.
Moreover, this approach can lead to an excess of ligands on the
nanoparticle surface, and this causes non-specific binding and
aggregation of nanoparticles, which decreases detection sensitivity.
Here, we show that magnetoferritin nanoparticles (M-HFn) can be used to
target and visualize tumour tissues without the use of any targeting
ligands or contrast agents. Iron oxide nanoparticles are encapsulated
inside a recombinant human heavy-chain ferritin (HFn) protein shell,
which binds to tumour cells that overexpress transferrin receptor 1
(TfR1). The iron oxide core catalyses the oxidation of peroxidase
substrates in the presence of hydrogen peroxide to produce a colour
reaction that is used to visualize tumour tissues. We examined 474
clinical specimens from patients with nine types of cancer and verified
that these nanoparticles can distinguish cancerous cells from normal
cells with a sensitivity of 98% and specificity of 95%.
Targeted delivery of chemotherapeutics is defined in the sense, that is, to maximize the therapeutic index of a chemotherapeutic agent by strictly localizing its pharmacological activity to the site or tissue of action. Integrins are a family of heterodimeric transmembrane glycoproteins involved in a wide range of cell-to-extracellular matrix (ECM) and cell-to-cell interactions. As cell surface receptors, integrins readily interact with extracellular ligands and play a vital role in angiogenesis, leukocytes function and tumor development, which sets up integrins as an excellent target for chemotherapy treatment. The peptide ligands containing the arginine-glycine-aspartic acid (RGD), which displays a strong binding affinity and selectivity to integrins, particularly to integrin αvβ3, have been developed to conjugate with various conventional chemotherapeutic agents, such as small molecules, peptides and proteins, and nanoparticle-carried drugs for integtrin targeted therapeutic studies. This review highlights the recent advances in integrin targeted delivery of chemotherapeutic agents with emphasis on target of integrin αvβ3, and describes the considerations for the design of the diverse RGD peptide-chemotherapeutics conjugates and their major applications.
Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated.
Optical imaging has emerged as a powerful modality for studying molecular recognitions and molecular imaging in a noninvasive, sensitive, and real-time way. Some advantages of optical imaging include cost-effectiveness, convenience, and non-ionization safety as well as complementation with other imaging modalities such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). Over the past decade, considerable advances have been made in tumor optical imaging by targeting integrin receptors in preclinical studies. This review has emphasized the construction and evaluation of diverse integrin targeting agents for optical imaging of tumors in mouse models. They mainly include some near-infrared fluorescent dye-RGD peptide conjugates, their multivalent analogs, and nanoparticle conjugates for targeting integrin αvβ3. Some compounds targeting other integrin subtypes such as α4β1 and α3 for tumor optical imaging have also been included. Both in vitro and in vivo studies have revealed some promising integrin-targeting optical agents which have further enhanced our understanding of integrin expression and targeting in cancer biology as well as related anticancer drug discovery. Especially, some integrin-targeted multifunctional optical agents including nanoparticle-based optical agents can multiplex optical imaging with other imaging modalities and targeted therapy, serving as an attractive type of theranostics for simultaneous imaging and targeted therapy. Continued efforts to discover and develop novel, innovative integrin-based optical agents with improved targeting specificity and imaging sensitivity hold great promises for improving cancer early detection, diagnosis, and targeted therapy in clinic.
Nanomaterials provide large surface areas, relativeto their volumes, on which to load functions. One challenge, however, has been to achieve precise control in loading multiple functionalities. Traditional bioconjugation techniques, which randomly target the surface functional groups of nanomaterials, have been found increasingly inadequate for such control, which is a drawback that may substantially slow down or prohibit the translational efforts. In the current study, we evaluated ferritin nanocages as candidate nanoplatforms for multifunctional loading. Ferritin nanocages can be either genetically or chemically modified to impart functionalities to their surfaces, and metal cations can be encapsulated in their interiors by association with metal binding sites. Moreover, different types of ferritin nanocages can be disassembled under acidic condition and reassembled at pH of 7.4, providing a facile way to achieve function hybridization. We were able to use combinations of these unique properties to produce a number of multifunctional ferritin nanostructures with precise control of their composition. We then studied these nanoparticles, both in vitro and in vivo, to evaluate their potential suitability as multimodality imaging probes. A good tumor targeting profile was observed, which was attributable to both the enhanced permeability and retention (EPR) effect and biovector mediated targeting. This, in combination with the generalizability of the function loading techniques, promises ferritin particles as a powerful nanoplatfom in the era of nanomedicine.
There is an opportunity to augment the therapeutic potential of drug combinations through use of drug delivery technology. This report summarizes data obtained using a novel liposomal formulation with coencapsulated doxorubicin and vincristine. The rationale for selecting these drugs is due in part to the fact that liposomal formulations of doxorubicin and vincristine are being separately evaluated as components of drug combinations.
Doxorubicin and vincristine were coencapsulated into liposomes using two distinct methods of drug loading. A manganese-based drug loading procedure, which relies on drug complexation with a transition metal, was used to encapsulate doxorubicin. Subsequently the ionophore A23187 was added to induce formation of a pH gradient, which promoted vincristine encapsulation.
Plasma elimination studies in mice indicated that the drug:drug ratio before injection [4:1 doxorubicin:vincristine (wt:wt ratio)] changed to 20:1 at the 24-h time point, indicative of more rapid release of vincristine from the liposomes than doxorubicin. Efficacy studies completed in MDA MB-435/LCC6 tumor-bearing mice suggested that at the maximum tolerated dose, the coencapsulated formulation was therapeutically no better than liposomal vincristine. This result was explained in part by in vitro cytotoxicity studies evaluating doxorubicin and vincristine combinations analyzed using the Chou and Talalay median effect principle. These data clearly indicated that simultaneous addition of vincristine and doxorubicin resulted in pronounced antagonism.
These results emphasize that in vitro drug combination screens can be used to predict whether a coformulated drug combination will act in an antagonistic or synergistic manner.
The dissociation of apoferritin into subunits at pH 2 followed by its reformation at pH 7.4 in presence of Desferrioxamine B (DFO) gives rise to a solution containing three DFO molecules trapped within the apoferritin (Apo-ferritin:DFO) and DFO molecules outside it. The untrapped DFO molecules in the solution were removed from Apo-ferritin:DFO by exhaustive dialysis until a negligible concentration was confirmed. The addition of Fe(III) to the dialyzed solution of Apo-ferritin:DFO resulted in the appearance of an orange-red color. The UV-Vis spectrum of this solution shows the characteristic absorption of the [DFOFe] complex centered at 425 nm. Following a similar procedure as for DFO, only one molecule of [DFOFe] was trapped in the apoferritin. The above results demonstrate the possibility of encapsulating a large molecule such as DFO in the apoferritin and, more interestingly, the ability of these DFO-encapsulated molecules to react with Fe(III) to give rise to an encapsulated [DFOFe] complex within the apoferritin.
Tumor vessel imaging could be useful in identifying angiogenic blood vessels as well as being a potential predictive marker of antiangiogenic treatment response. We recently reported the expression of the neural cell adhesion molecule (NCAM) in the immature and tumor endothelial cell (TEC) lining vessels of human carcinomas. Exploiting an in vivo model of human tumor angiogenesis obtained by implantation of TEC in Matrigel in severe combined immunodeficiency mice, we aimed to image angiogenesis by detecting the expression of NCAM with magnetic resonance imaging. The imaging procedure consisted of (a) targeting NCAMs with a biotinylated derivative of C3d peptide that is known to have high affinity for these epitopes and (b) delivery of a streptavidin/gadolinium (Gd)-loaded apoferritin 1:1 adduct at the biotinylated target sites. The remarkable relaxation enhancement ability of the Gd-loaded apoferritin system allowed the visualization of TEC both in vitro and in vivo when organized in microvessels connected to the mouse vasculature. Gd-loaded apoferritin displayed good in vivo stability and tolerability. The procedure reported herein may be easily extended to the magnetic resonance visualization of other epitopes suitably targeted by proper biotinylated vectors.
The chemical construction of dipersed CdS nanoparticles within the polypeptide cage of apoferritin is demonstrated to be possible by a multistep synthetic route, as shown schematically in the Figure. Ferritin-induced nucleation of CdS clusters (squares) is followed by reaction with excess sulfide to give CdS-ferritin. Such biocompatible nanoparticles might find uses as tunable photochemical agents for diagnosis, sensing, and drug delivery.
The formation constants for complexes formed between Adriamycin and some transition metals are reported. They are used to calculate the probable complex species concentrations present in gastro-intestinal fluids following Adriamycin and Quelamycin administration and to estimate the influence of the two agents upon the low-molecular-weight complexes normally present in blood plasma. Adriamycin forms a range of complexes in intestinal fluid and blood plasma, e.g. CaAd and FeAdOH. The former suggests that Adriamycin may affect calcium metabolism whereas the latter suggests that co-administering ferric ions could interfere with this inhibition.
Engineered nanoparticles have been used to provide diagnostic, therapeutic and prognostic information about the status of disease. Nanoparticles developed for these purposes are typically modified with targeting ligands (such as antibodies, peptides or small molecules) or contrast agents using complicated processes and expensive reagents. Moreover, this approach can lead to an excess of ligands on the nanoparticle surface, and this causes non-specific binding and aggregation of nanoparticles, which decreases detection sensitivity. Here, we show that magnetoferritin nanoparticles (M-HFn) can be used to target and visualize tumour tissues without the use of any targeting ligands or contrast agents. Iron oxide nanoparticles are encapsulated inside a recombinant human heavy-chain ferritin (HFn) protein shell, which binds to tumour cells that overexpress transferrin receptor 1 (TfR1). The iron oxide core catalyses the oxidation of peroxidase substrates in the presence of hydrogen peroxide to produce a colour reaction that is used to visualize tumour tissues. We examined 474 clinical specimens from patients with nine types of cancer and verified that these nanoparticles can distinguish cancerous cells from normal cells with a sensitivity of 98% and specificity of 95%.
An ongoing effort in the field of nanomedicine is to develop nanoplatforms with both imaging and therapeutic functions, the "nanotheranostics". We have previously developed a human serum albumin (HSA) coated iron oxide nanoparticle (HINP) formula and used multiple imaging modalities to validate its tumor targeting attributes. In the current study, we sought to impart doxorubicin (Dox) onto the HINPs and to assess the potential of the conjugates as theranostic agents. In a typical preparation, we found that about 0.5 mg of Dox and 1 mg of iron oxide nanoparticles (IONPs, Fe content) could be loaded into 10 mg of HSA matrices. The resulting D-HINPs (Dox loaded HINPs) have a hydrodynamic size of 50 nm and are able to release Dox in a sustained fashion. More impressively, the HINPs can assist the translocation of Dox across the cell membrane and even its accumulation in the nucleus. In vivo, D-HINPs retained a tumor targeting capability of HINPs, as manifested by both in vivo MRI and ex vivo immunostaining results. In a follow-up therapeutic study on a 4T1 murine breast cancer xenograft model, D-HINPs showed a striking tumor suppression effect that was comparable to that of Doxil and greatly outperformed free Dox. Such a strategy can be readily extended to load other types of small molecules, making HINP a promising theranostic nanoplatform.
Molecular imaging is a key component of 21st-century cancer management. The vascular endothelial growth factor (VEGF)/VEGF receptor signaling pathway and integrin alpha v beta 3, a cell adhesion molecule, play pivotal roles in regulating tumor angiogenesis, the growth of new blood vessels. This review summarizes the current status of tumor angiogenesis imaging with SPECT, PET, molecular MRI, targeted ultrasound, and optical techniques. For integrin alpha v beta 3 imaging, only nanoparticle-based probes, which truly target the tumor vasculature rather than tumor cells because of poor extravasation, are discussed. Once improvements in the in vivo stability, tumor-targeting efficacy, and pharmacokinetics of tumor angiogenesis imaging probes are made, translation to clinical applications will be critical for the maximum benefit of these novel agents. The future of tumor angiogenesis imaging lies in multimodality and nanoparticle-based approaches, imaging of protein-protein interactions, and quantitative molecular imaging. Combinations of multiple modalities can yield complementary information and offer synergistic advantages over any modality alone. Nanoparticles, possessing multifunctionality and enormous flexibility, can allow for the integration of therapeutic components, targeting ligands, and multimodality imaging labels into one entity, termed "nanomedicine," for which the ideal target is tumor neovasculature. Quantitative imaging of tumor angiogenesis and protein-protein interactions that modulate angiogenesis will lead to more robust and effective monitoring of personalized molecular cancer therapy. Multidisciplinary approaches and cooperative efforts from many individuals, institutions, industries, and organizations are needed to quickly translate multimodality tumor angiogenesis imaging into multiple facets of cancer management. Not limited to cancer, these novel agents can also have broad applications for many other angiogenesis-related diseases.
To address two fundamental and unsolved problems in optical imaging (nonspecific uptake of near-infrared fluorophores by normal tissues and organs and incomplete elimination of unbound targeted fluorophores from the body), novel zwitterionic near-infrared fluorophores (e.g., ZW800-1) were synthesized and their performance compared in vivo to conventional molecules (e.g., ICG) as a function of charge, charge distribution, and hydrophobicity (see picture).
Optical imaging has emerged as a powerful modality for studying molecular recognitions and molecular imaging in a noninvasive, sensitive, and real-time way. Some advantages of optical imaging include cost-effectiveness, convenience, and non-ionization safety as well as complementation with other imaging modalities such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). Over the past decade, considerable advances have been made in tumor optical imaging by targeting integrin receptors in preclinical studies. This review has emphasized the construction and evaluation of diverse integrin targeting agents for optical imaging of tumors in mouse models. They mainly include some near-infrared fluorescent dye-RGD peptide conjugates, their multivalent analogs, and nanoparticle conjugates for targeting integrin αvβ3. Some compounds targeting other integrin subtypes such as α4β1 and α3 for tumor optical imaging have also been included. Both in vitro and in vivo studies have revealed some promising integrin-targeting optical agents which have further enhanced our understanding of integrin expression and targeting in cancer biology as well as related anticancer drug discovery. Especially, some integrin-targeted multifunctional optical agents including nanoparticle-based optical agents can multiplex optical imaging with other imaging modalities and targeted therapy, serving as an attractive type of theranostics for simultaneous imaging and targeted therapy. Continued efforts to discover and develop novel, innovative integrin-based optical agents with improved targeting specificity and imaging sensitivity hold great promises for improving cancer early detection, diagnosis, and targeted therapy in clinic.
Functional nanostructures with high biocompatibility and stability, low toxicity, and specificity of targeting to desired organs or cells are of great interest in nanobiology and medicine. However, the challenge is to integrate all of these desired features into a single nanobiostructure, which can be applied to biomedical applications and eventually in clinical settings. In this context, we designed a strategy to assemble two gold nanoclusters at the ferroxidase active sites of ferritin heavy chain. Our studies showed that the resulting nanostructures (Au-Ft) retain not only the intrinsic fluorescence properties of noble metal, but gain enhanced intensity, show a red-shift, and exhibit tunable emissions due to the coupling interaction between the paired Au clusters. Furthermore, Au-Ft possessed the well-defined nanostructure of native ferritin, showed organ-specific targeting ability, high biocompatibility, and low cytotoxicity. The current study demonstrates that an integrated multimodal assembly strategy is able to generate stable and effective biomolecule-noble metal complexes of controllable size and with desirable fluorescence emission characteristics. Such agents are ideal for targeted in vitro and in vivo imaging. These results thus open new opportunities for biomolecule-guided nanostructure assembly with great potential for biomedical applications.
Rattling the cage: Protein cages are used as a scaffold to build protease-activatable probes. The self-assembly of ferritin cages generates hybrid proteins with matrix metalloproteinase (MMP)-specific activation (see picture). The formula with the highest activation efficiency is validated as a tumor-specific probe in a xenograft mouse model.
Repeated administration of chemotherapeutics is typically required for the effective treatment of highly aggressive tumors and often results in systemic toxicity. We have created a copper-doxorubicin complex within the core of liposomes and applied the resulting particle in multidose therapy. Copper and doxorubicin concentrations in the blood pool were similar at 24 h (∼40% of the injected dose), indicating stable circulation of the complex. Highly quenched doxorubicin fluorescence remained in the blood pool over tens of hours, with fluorescence increasing only with the combination of liposome disruption and copper trans-chelation. At 48 h after injection, doxorubicin fluorescence within the heart and skin was one-fifth and one-half, respectively, of fluorescence observed with ammonium sulfate-loaded doxorubicin liposomes. After 28 days of twice per week doxorubicin administration of 6 mg/kg, systemic toxicity (cardiac hypertrophy and weight and hair loss) was not detected with the copper-doxorubicin liposomes but was substantial with ammonium sulfate-loaded doxorubicin liposomes. We then incorporated two strategies designed to enhance efficacy, mTOR inhibition (rapamycin) to slow proliferation and therapeutic ultrasound to enhance accumulation and local diffusion. Tumor accumulation was ∼10% ID/g and was enhanced approximately 2-fold with the addition of therapeutic ultrasound. After the 28-day course of therapy, syngeneic tumors regressed to a premalignant phenotype of ∼(1 mm)(3) or could not be detected.
Numerous peptide-based molecular imaging probes with respect to their design strategies and applications are studied. Somatostatin (SST), integrin, gastrin-releasing peptide (GRP), cholecystokinin (CCK), R-melanocyte stimulating hormone (R-MSH), and glucagon-like peptide-1 (GLP-1) receptors have been identified and characterized for tumor receptor imaging. An imaging probe can be created through combination of a targeting peptide, a linker, and an imaging moiety. The new peptide associated probe should have high in ViVo uptake and retention in the target, with low background uptake in non-target tissues. Despite the rapid progress of a number of imaging modalities, nuclear imaging remains the premier clinical method. Both PET and SPECT have been well-developed and are widely used in daily practice. A critical step in the development of peptide probes for nuclear imaging is the radiolabeling process. Real-time imaging of biological processes in live cells and in vivo is one of the basic goals of modern optical imaging techniques.
A key challenge in developing nanoplatform-based molecular imaging is to achieve an optimal pharmacokinetic profile to allow sufficient targeting and to avoid rapid clearance by the reticuloendothelial system (RES). In the present study, iron oxide nanoparticles (IONPs) were coated with a PEGylated amphiphilic triblock copolymer, making them water soluble and function-extendable. These particles were then conjugated with a near-infrared fluorescent (NIRF) dye IRDye800 and cyclic Arginine-Glycine-Aspartic acid (RGD) containing peptide c(RGDyK) for integrin alpha(v)beta(3) targeting. In vitro binding assays confirmed the integrin-specific association between the RGD-particle adducts and U87MG glioblastoma cells. Successful tumor homing in vivo was perceived in a subcutaneous U87MG glioblastoma xenograft model by both magnetic resonance imaging (MRI) and NIRF imaging. Ex vivo histopathological studies also revealed low particle accumulation in the liver, which was attributed to their compact hydrodynamic size and PEGylated coating. In conclusion, we have developed a novel RGD-IONP conjugate with excellent tumor integrin targeting efficiency and specificity as well as limited RES uptake for molecular MRI.
Protein‐based nanomedicine platforms for drug delivery comprise naturally self‐assembled protein subunits of the same protein or a combination of proteins making up a complete system. They are ideal for drug‐delivery platforms due to their biocompatibility and biodegradability coupled with low toxicity. A variety of proteins have been used and characterized for drug‐delivery systems, including the ferritin/apoferritin protein cage, plant‐derived viral capsids, the small Heat shock protein (sHsp) cage, albumin, soy and whey protein, collagen, and gelatin. There are many different types and shapes that have been prepared to deliver drug molecules using protein‐based platforms, including various protein cages, microspheres, nanoparticles, hydrogels, films, minirods, and minipellets. The protein cage is the most newly developed biomaterial for drug delivery and therapeutic applications. The uniform size, multifunctionality, and biodegradability push it to the frontier of drug delivery. In this Review, the recent strategic development of drug delivery is discussed with emphasis on polymer‐based, especially protein‐based, nanomedicine platforms for drug delivery. The advantages and disadvantages are also discussed for each type of protein‐based drug‐delivery system.
magnified image
Molecular imaging has evolved over the past several years into an important tool for diagnosing, understanding, and monitoring disease. Molecular imaging has distinguished itself as an interdisciplinary field, with contributions from chemistry, biology, physics, and medicine. The cross-disciplinary impetus has led to significant achievements, such as the development of more sensitive imaging instruments and robust, safer radiopharmaceuticals, thereby providing more choices to fit personalized medical needs. Molecular imaging is making steadfast progress in the field of cancer research among others. Cancer is a challenging disease, characterized by heterogeneity, uncontrolled cell division, and the ability of cancer cells to invade other tissues. Researchers are addressing these challenges by aggressively identifying and studying key cancer-specific biomarkers such as growth factor receptors, protein kinases, cell adhesion molecules, and proteases, as well as cancer-related biological processes such as hypoxia, apoptosis, and angiogenesis.
The cardiotoxic effects of adriamycin were studied in 399 patients treated for far-advanced carcimma. Forty-five patients (11%) exhibited transient electrocardiographs changes. Eleven others developed severe congestive heart failure. Eight of these latter patients died within 3 weeks of the onset of the cardiac decompensation. The diffuse nature of this myocardiopathy was suggested by: 1. a conspicuous decrease in the QRS voltage on the electrocardiograms; 2. rapidly occurring cardiac dilatation and ventricular failure, and 3. refractoriness to inoiropic drugs and mechanical ventricular assistance. Postmortem examination of the hearts in two cases showed a striking decrease in the number of cardiac: muscle cells present, degeneration of the remaining myocardial cells, loss o contractile substance, mitochondrial swelling, and intramitochondrial dense inclusion bodies. Congestive heart failure occurred only once in the 366 patients who were treated with less than 550 mg/m2 of adriamycin (0.27%), but there were 10 cases of cardiac failure in the 33 patients who received more than 550 mg/m2 of this drug (30%). Therefore, until more direct means are established to prevent adriamycin-induced congestive heart failure, it is suggested that the total dose of adriamycin should be limited to less than 550 mg/m2 to permit safer use of this efficacious cancer chemotherapeutic agent.
The complexes of adriamycin (ADM) with Cu(II) and Fe(II) have been studied by visible absorption, circular dichroism (CD) and fluorescence spectra, respectively. In Tris buffer at pH 7.0, either metal ions forms a single species with adriamycin: Cu(ADM)2 or Fe(ADM)3. Interaction of these two complexes with various biological molecules has been examined. It is shown that some amino acids, glutathione and albumin are able to remove the Cu(II) ion from Cu(II)-ADM complex, releasing the free drug. However, Fe(II)-ADM keeps in an undissociated form under the same conditions. The possibility of Fe(II) ADM as a new alternative drug has been discussed.
The empty protein cage of ferritin has been used to synthesize and entrap nanoscale particles of a green cobalt oxide via oxidative hydrolysis of Co(II) by H 2O 2. The resulting composite material retains the properties of the protein while incorporating the characteristics of the encapsulated mineral. The visible absorption spectrum has a broad band at 350 nm and the IR spectrum shows a band at 587 cm -1 characteristic of the cobalt oxyhydroxide Co(O)OH.
The iron storage protein ferritin contains threefold and fourfold symmetric channels that are thought to provide pathways for the transfer of Fe(2+) ions in and out of the protein. Using the known crystal structure of the ferritin protein, we perform electrostatic potential energy calculations to elucidate the functional properties of these channels. The threefold channel is shown to be responsible for the transit of Fe(2+) ions. Monovalent ions can also diffuse through the threefold channel but presence of divalent ions in the pore retards this process leading to a selectivity mechanism similar to the one observed in calcium channels. The fourfold channel is found to be impermeant to all cations with the possible exception of protons. Because proton transfer is essential to maintain the electroneutrality of the protein during iron deposition, we suggest that the function of the fourfold channel is to form a "proton wire" that facilitates their transfer in and out of ferritin.
We report the in vivo targeting and imaging of tumor vasculature using arginine-glycine-aspartic acid (RGD) peptide-labeled quantum dots (QDs). Athymic nude mice bearing subcutaneous U87MG human glioblastoma tumors were administered QD705-RGD intravenously. The tumor fluorescence intensity reached maximum at 6 h postinjection with good contrast. The results reported here open up new perspectives for integrin-targeted near-infrared optical imaging and may aid in cancer detection and management including imaging-guided surgery.
Protein cage architectures such as virus capsids and ferritins are versatile nanoscale platforms amenable to both genetic and chemical modification. Incorporation of multiple functionalities within these nanometer-sized protein architectures demonstrate their potential to serve as functional nanomaterials with applications in medical imaging and therapy. In the present study, we synthesized an iron oxide (magnetite) nanoparticle within the interior cavity of a genetically engineered human H-chain ferritin (HFn). A cell-specific targeting peptide, RGD-4C which binds alphavbeta3 integrins upregulated on tumor vasculature, was genetically incorporated on the exterior surface of HFn. Both magnetite-containing and fluorescently labeled RGD4C-Fn cages bound C32 melanoma cells in vitro. Together these results demonstrate the capability of a genetically modified protein cage architecture to serve as a multifunctional nanoscale container for simultaneous iron oxide loading and cell-specific targeting.
Apoferritin derived from the native protein ferritin was employed to encapsulate anticancer drugs cisplatin and carboplatin.
The cell adhesion molecule integrin alpha vbeta 3 plays a key role in tumor angiogenesis and metastasis. A series of (18)F-labeled RGD peptides have been developed for PET of integrin expression based on primary amine reactive prosthetic groups. In this study, we report the use of the Cu(I)-catalyzed Huisgen cycloaddition, also known as a click reaction, to label RGD peptides with (18)F by forming 1,2,3-triazoles. Nucleophilic fluorination of a toluenesulfonic alkyne provided (18)F-alkyne in high yield (nondecay-corrected yield: 65.0 +/- 1.9%, starting from the azeotropically dried (18)F-fluoride), which was then reacted with an RGD azide (nondecay-corrected yield: 52.0 +/- 8.3% within 45 min including HPLC purification). The (18)F-labeled peptide was subjected to microPET studies in murine xenograft models. Murine microPET experiments showed good tumor uptake (2.1 +/- 0.4%ID/g at 1 h postinjection (p.i.)) with rapid renal and hepatic clearance of (18)F-fluoro-PEG-triazoles-RGD 2 ( (18)F-FPTA-RGD2) in a subcutaneous U87MG glioblastoma xenograft model (kidney 2.7 +/- 0.8%ID/g; liver 1.9 +/- 0.4%ID/g at 1 h p.i.). Metabolic stability of the newly synthesized tracer was also analyzed (intact tracer ranging from 75% to 99% at 1 h p.i.). In brief, the new tracer (18)F-FPTA-RGD2 was synthesized with high radiochemical yield and high specific activity. This tracer exhibited good tumor-targeting efficacy and relatively good metabolic stability, as well as favorable in vivo pharmacokinetics. This new (18)F labeling method based on click reaction may also be useful for radiolabeling of other biomolecules with azide groups in high yield.
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