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Magnetoferritin nanoparticles for targeting and visualizing tumour tissues
Abstract
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%.
... Moreover, this approach is economic, rapid, and simple. For tumor detection, it can simplify the conventional immunohistochemical method and reduce the test time from 5 to 1 hour and improve the effectiveness of the diagnostic test 95 (Table 3). ...
... A strategy was developed for concurrently focusing on and picturing tumors based on the inborn peroxidase-like movement of iron oxide NPs. 95 Iron oxide nanoparticles (NPs) can be identified by human overwhelming chain ferritin (HFn). The target of the specific official of HFn to this receptor is the transferrin receptor 1 (TfR 1), which is overexpressed in malignant cells. ...
... The magnetoferritin-NP complex may offer an effective alternative to conventional cancer and tumour treatment. 95 Under physiological conditions, peroxidase-active iron oxide nanoparticles (NPs) deliver oxygen radicals close to H 2 O 2 based on the Fenton or Haber-Weiss reaction. 81,132 Germs and cancerous cells are destroyed by oxidative stress, which is brought on by an increase in ROS. ...
Breast and ovarian cancers, despite having chemotherapy and surgical treatment, still have the lowest survival rate. Experimental stages using nanoenzymes/nanozymes for ovarian cancer diagnosis and treatment are being carried out, and correspondingly the current treatment approaches to treat breast cancer have a lot of adverse side effects, which is the reason why researchers and scientists are looking for new strategies with less side effects. Nanoenzymes have intrinsic enzyme-like activities and can reduce the shortcomings of naturally occurring enzymes due to the ease of storage, high stability, less expensive, and enhanced efficiency. In this review, we have discussed various ways in which nanoenzymes are being used to diagnose and treat breast and ovarian cancer. For breast cancer, nanoenzymes and their multi-enzymatic properties can control the level of reactive oxygen species (ROS) in cells or tissues, for example, oxidase (OXD) and peroxidase (POD) activity can be used to generate ROS, while catalase (CAT) or superoxide dismutase (SOD) activity can scavenge ROS. In the case of ovarian cancer, most commonly nanoceria is being investigated, and also when folic acid is combined with nanoceria there are additional advantages like inhibition of beta galactosidase. Nanocarriers are also used to deliver small interfering RNA that are effective in cancer treatment. Studies have shown that iron oxide nanoparticles are actively being used for drug delivery, similarly ferritin carriers are used for the delivery of nanozymes. Hypoxia is a major factor in ovarian cancer, therefore MnO2-based nanozymes are being used as a therapy. For cancer diagnosis and screening, nanozymes are being used in sonodynamic cancer therapy for cancer diagnosis and screening, whereas biomedical imaging and folic acid gold particles are also being used for image guided treatments. Nanozyme biosensors have been developed to detect ovarian cancer. This review article summarizes a detailed insight into breast and ovarian cancers in light of nanozymes-based diagnostic and therapeutic approaches.
... The interior cavity of ferritin protein cage has been extensively employed as a natural template for preparing highly monodisperse and water-soluble metallic nanoparticles (NPs) [10][11][12][13]. We have previously reported that human HFn could specifically accumulate into tumors and atherosclerotic plaques via transferrin receptor 1 (TfR1)-mediated specific targeting followed by rapid internalization [14][15][16][17][18][19]. Herein, we synthesized a highly monodisperse Gd core within the interior cavity of HFn nanocage and achieved a sensitive MRI tracking of TfR1 with a remarkable enhanced relaxivity of water protons. ...
... Recombinant human HFn nanocages were expressed and purified from E. coli, as descried previously [14]. Gd-HFn NPs were prepared by loading Gd 3+ into the cavities of HFn nanocages through the ion channel at the threefold axis of HFn nanocage, followed by the formation of Gd nanoparticle within the nucleation site ( Figure 1A). ...
... We have previously reported that HFn protein can selectively bind to human tumors and significantly accumulate into tumor cells via TfR1-mediated specific targeting and the subsequent robust internalization [14,15]. The prepared HFn nanocages thus should be able to deliver the confined Gd NPs into tumor cells and perform Gd-based tumor MR imaging. ...
Rationale: Magnetic resonance imaging (MRI) is a powerful diagnostic technology by providing high-resolution imaging. Although MRI is sufficiently valued in its resolving morphology, it has poor sensitivity for tracking biomarkers. Therefore, contrast agents are often used to improve MRI diagnostic sensitivity. However, the clinically used Gd chelates are limited in improving MRI sensitivity owing to their low relaxivity. The objective of this study is to develop a novel contrast agent to achieve a highly sensitive tracking of biomarkers in vivo.
Methods: A Gd-based nanoprobe composed of a gadolinium nanoparticle encapsulated within a human H-ferritin nanocage (Gd-HFn) has been developed. The specificity and sensitivity of Gd-HFn were evaluated in vivo in tumor-bearing mice and apolipoprotein E-deficient mice (Apoe-/-) by MRI.
Results: The Gd-HFn probe shows extremely high relaxivity values (r1 = 549 s⁻¹mM⁻¹, r2 = 1555 s⁻¹mM⁻¹ under a 1.5-T magnetic field; and r1 = 428 s⁻¹mM⁻¹ and r2 = 1286 s⁻¹mM⁻¹ under a 3.0-T magnetic field), which is 175-fold higher than that of the clinically standard Dotarem (Gd-DOTA, r1 =3.13 s⁻¹mM⁻¹) under a 1.5-T magnetic field, and 150-fold higher under a 3.0-T magnetic field. Owing to the substantially enhanced relaxivity values, Gd-HFn achieved a highly sensitive tracking for the tumor targeting receptor of TfR1 and enabled the in vivo MRI visualization of tumors approaching the angiogenic switch.
Conclusions: The developed Gd-HFn contrast agent makes MRI a more powerful tool by simultaneously providing functional and morphological imaging information, which paves the way for a new perspective in molecular imaging.
... Here, DOX is encapsulated within a 12 nm diameter cave sphere constituted by 24 HFn subunits [23]. This nanoplatform is able to specifically recognize the TfR1, which is highly expressed in cancer tissues and mediates the internalization of nanoparticles, thus facilitating the tumor-targeted recognition of this nanoformulation [40]. Thanks to its unique biotechnological properties, HFn quaternary structure could be disassembled lowering the pH until 2.0, and then refolded bringing back the pH to the neutrality [19,23]. ...
... On the other side, both FerOX and free DOX exhibited greater efficacy than Myocet in inhibiting the proliferation of BC-PDO, as shown by the nearly 15-to 20-fold increase in Myocet IC 50 (Fig. 2B). Moreover, FerOX IC 50 values displayed an inverse correlation with TfR1 expression, confirming the capability of FerOX to mediate a specific tumor-targeted delivery of DOX, as previously described in literature (Fig. 4C) [19,23,24,36,40]. ...
Despite the advent of numerous targeted therapies in clinical practice, anthracyclines, including doxorubicin (DOX), continue to play a pivotal role in breast cancer (BC) treatment. DOX directly disrupts DNA replication, demonstrating remarkable efficacy against BC cells. However, its non-specificity toward cancer cells leads to significant side effects, limiting its clinical utility. Interestingly, DOX can also enhance the antitumor immune response by promoting immunogenic cell death in BC cells, thereby facilitating the presentation of tumor antigens to the adaptive immune system. However, the generation of an adaptive immune response involves highly proliferative processes, which may be adversely affected by DOX-induced cytotoxicity. Therefore, understanding the impact of DOX on dividing T cells becomes crucial, to deepen our understanding and potentially devise strategies to shield anti-tumor immunity from DOX-induced toxicity. Our investigation focused on studying DOX uptake and its effects on human lymphocytes. We collected lymphocytes from healthy donors and BC patients undergoing neoadjuvant chemotherapy (NAC). Notably, patient-derived peripheral blood mononuclear cells (PBMC) promptly internalized DOX when incubated in vitro or isolated immediately after NAC. These DOX-treated PBMCs exhibited significant proliferative impairment compared to untreated cells or those isolated before treatment initiation. Intriguingly, among diverse lymphocyte sub-populations, CD8 + T cells exhibited the highest uptake of DOX. To address this concern, we explored a novel DOX formulation encapsulated in ferritin nanocages (FerOX). FerOX specifically targets tumors and effectively eradicates BC both in vitro and in vivo. Remarkably, only T cells treated with FerOX exhibited reduced DOX internalization, potentially minimizing cytotoxic effects on adaptive immunity.
Our findings underscore the importance of optimizing DOX delivery to enhance its antitumor efficacy while minimizing adverse effects, highlighting the pivotal role played by FerOX in mitigating DOX-induced toxicity towards T-cells, thereby positioning it as a promising DOX formulation. This study contributes valuable insights to modern cancer therapy and immunomodulation.
... TRFR1 is expressed at low levels in normal cells but has an abnormal overexpression state in common cancers, such as BC and lung cancers [25,26]. Hence, heavy-chain ferritin vehicles are tumor-selective without the addition of targeting ligands [27]. 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]. ...
... TRFR1 is expressed at low levels in normal cells but has an abnormal overexpression state in common cancers, such as BC and lung cancers [25,26]. Hence, heavy-chain ferritin vehicles are tumor-selective without the addition of targeting ligands [27]. 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]. ...
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.
... ApoA-I Milano/phospholipids weekly for 5 weeks reduced coronary atherosclerosis [58]. [59]. HFn-Dox bound and internalised tumour cells via overexpressed TfR1 and released Dox in the lysosomes. ...
This book series invites all the Specialists, Professors, Doctors, Scientists, Academicians, Healthcare professionals, Nurses, Students, Researchers, Business Delegates, and Industrialists across the globe to publish their insights and convey recent developments in the field of Nursing, Pharmaceutical Research and Innovations in Pharma Industry. Book series on Pharmacy and Nursing covers research work in a set of clinical sciences and medicine.
... AML is a hematologic malignancy in which the PD-L1 level is upregulated, and therefore, inhibition of the PD-1/PD-L1 axis is associated with a better treatment outcome [23]. On the other hand, studies reported that HFn can specifically bind CD71-overexpressing cells [11,24]. Here, we conducted RT-PCR to evaluate the silencing potency of siPD-L1/HFn. ...
Because of the high biocompatibility, self-assembly capability, and CD71-mediated endocytosis, using human heavy chain ferritin (HFn) as a nanocarrier would greatly increase therapeutic effectiveness and reduce possible adverse events. Anti-PD-L1 siRNA can downregulate the level of PD-L1 on tumor cells, resulting in the activation of effector T cells against leukemia. Therefore, this study aimed to produce the tumor-targeting siPD-L1/HFn nanocarrier. Briefly, the HFn coding sequence was cloned into a pET-28a, and the constructed expression plasmid was subsequently transformed into E. coli BL21. After induction of Isopropyl β-d-1-thiogalactopyranoside (IPTG), HFn was purified with Ni-affinity chromatography and dialyzed against PBS. The protein characteristics were analyzed using SDS-PAGE, Western Blot, and Dynamic light scattering (DLS). The final concentration was assessed using the Bicinchoninic acid (BCA) assay. The encapsulation was performed using the standard pH system. The treatment effects of siPD-L1/HFn were carried out on HL-60 and K-562 cancer cell lines. The RT-PCR was used to determine the mRNA expression of PD-L1. The biocompatibility and excretion of siPD-L1/HFn have also been evaluated. The expression and purity of HFn were well verified through SDS-PAGE, WB, and DLS. RT-PCR analyses also showed significant siRNA-mediated PD-L1 silencing in both HL-60 and K-562 cells. Our study suggested a promising approach for siRNA delivery. This efficient delivery system can pave the way for the co-delivery of siRNAs and multiple chemotherapies to address the emerging needs of cancer combination therapy.
... Mn(II) was loaded into the nanocage of apoferritin, and further oxidized at pH 9, followed by the formation of Mn(III) oxyhydroxide nanoparticles with enhanced T1-weighted MRI capability in vivo (Meldrum et al., 1995, Geninatti Crich et al., 2012. Iron-oxide nanoparticles have also been synthesized in the hollow cage of recombinant human ferritin to achieve magnetoferritin nanoparticles for visualizing tumors via peroxidase activity of iron oxide (Fan et al., 2012;Gao et al., 2007). In order to distinguish malignant cells from healthy cells with the means of color response, the iron-oxide core can accelerate the oxidation of peroxidase targets in the presence of hydrogen peroxide, showing a great potential for rapid, low-cost, and universal tumor diagnosis. ...
The clinical advancement of protein-based nanomedicine has revolutionized medical professionals' perspectives on cancer therapy. Protein-based nanoparticles have been exploited as attractive vehicles for cancer nanomedicine due to their unique properties derived from naturally biomacromolecules with superior biocompatibility and pharmaceutical features. Furthermore, the successful translation of Abraxane™ (paclitaxel-based albumin nanoparticles) into clinical application opened a new avenue for protein-based cancer nanomedicine. In this mini-review article, we demonstrate the rational design and recent progress of protein-based nanoparticles along with their applications in cancer diagnosis and therapy from recent literature. The current challenges and hurdles that hinder clinical application of protein-based nanoparticles are highlighted. Finally, future perspectives for translating protein-based nanoparticles into clinic are identified.
... 8,9,14,15 ISL has been used to gain structural information on biological molecules (i.e., large proteins and viruses) due to its ability to largely preserve biological activity and conformations. 16,17 For example, in biomedical applications such as the synthesis and coating of particles and proteins used for disease diagnosis 18 and drug delivery vehicles, 19,20 high selectivity enabled by mass selection alone or in combination with structural separations prior to deposition plays a key role in developing an understanding at the molecular level. In combination with rapidly advancing ultrahigh-resolution imaging technologies (i.e., cryo-EM, 13 low-energy electron holography, 21 scanning probe microscopy, 22 and scanning tunneling microscopy 23 ) ISL has recently been shown as an emergent approach to enable the characterization of molecular structures at the single-molecule level. ...
While conventional ion-soft landing uses the massto-
charge (m/z) ratio to achieve molecular selection for deposition,
here we demonstrate the use of Structures for Lossless Ion
Manipulation (SLIM) for mobility-based ion selection and
deposition. The dynamic rerouting capabilities of SLIM were
leveraged to enable the rerouting of a selected range of mobilities
to a different SLIM path (rather than MS) that terminated at a
deposition surface. A selected mobility range from a phosphazene
ion mixture was rerouted and deposited with a current pulse (∼150
pA) resembling its mobility peak. In addition, from a mixture of
tetra-alkyl ammonium (TAA) ions containing chain lengths of
C5−C8, selected chains (C6, C7) were collected on a surface,
reconstituted into solution-phase, and subsequently analyzed with
a SLIM-qToF to obtain an IMS/MS spectrum, confirming the identity of the selected species. Further, this method was used to
characterize triply charged tungsten-polyoxometalate anions, PW12O40
3− (WPOM). The arrival time distribution of the IMS/MS
showed multiple peaks associated with the triply charged anion (PW12O40
3−), of which a selected ATD was deposited and imaged
using TEM. Additionally, the identity of the deposited WPOM was ascertained using energy-dispersive (EDS) spectroscopy.
Further, we present theory and computations that reveal ion landing energies, the ability to modulate the energies, and deposition
... In particular, human heavy chain ferritin (HFn) has garnered attention as an effective drug delivery carrier for cancer therapy [10][11][12][13] . Through its specific binding to transferrin receptor 1 (TfR1), ferritin can specifically target cancer cells [14][15][16] . Ferritin, a conserved protein renowned for its iron binding and storage capabilities, possesses a self-assembling structure comprised of a hollow protein shell and this shell exhibits an outer diameter measuring 12 nm and an inner diameter measuring 8 nm [17][18][19][20] . ...
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.
... With the high stability and easy modification of ferritin structures, the finding of the SOD-like activity of natural ferritin nanozyme would not only contribute to the understanding of the biominerals but also offer a new strategy for the superoxide-related disease. For example, our previous works have proved that HFn itself could target the tumor cells with high specificity and sensitivity based on their TfR1 binding ability 62 . In addition, the overexpression of superoxide dismutase has been found to inhibit the growth or invasion of the tumor cells 63,64 . ...
Biominerals, the inorganic minerals of organisms, are known mainly for their physical property-related functions in modern living organisms. Our recent discovery of the enzyme-like activities of nanomaterials, coined as nanozyme, inspires the hypothesis that nano-biominerals might function as enzyme-like catalyzers in cells. Here we report that the iron cores of biogenic ferritins act as natural nanozymes to scavenge superoxide radicals. Through analyzing eighteen representative ferritins from three living kingdoms, we find that the iron core of prokaryote ferritin possesses higher superoxide-diminishing activity than that of eukaryotes. Further investigation reveals that the differences in catalytic capability result from the iron/phosphate ratio changes in the iron core, which is mainly determined by the structures of ferritins. The phosphate in the iron core switches the iron core from single crystalline to amorphous iron phosphate-like structure, resulting in decreased affinity to the hydrogen proton of the ferrihydrite-like core that facilitates its reaction with superoxide in a manner different from that of ferric ions. Furthermore, overexpression of ferritins with high superoxide-diminishing activities in E. coli increases the resistance to superoxide, whereas bacterioferritin knockout or human ferritin knock-in diminishes free radical tolerance, highlighting the physiological antioxidant role of this type of nanozymes.
... Hydrophobic drugs may be attached by the π-π stacking mechanism to improve dispersion and facilitate release of the drugs and genes [69][70][71] . A few targeting molecules to deliver selectively to cancerous cells may be anchored through click chemistry or covalent modifications [72][73][74][75] . Biological molecules such as proteins, peptides and nucleic acid may be anchored through electrostatic attraction, click chemistry or covalent interactions to enable ameliorated biocompatibility, solubility and enhanced shielding from uncontrolled degradation and cleavage 69,70 . ...
Cancer, a disease with complex pathological process, is the principal cause of mortality and morbidity throughout the world. Conventional chemotherapy faces drawbacks regarding its low bioavaiability, insolubility, high dose requirements, low therapeutic indices, cytotoxicity, multiple drug resistance-development, biological barriers related obstruction, and non-specific targeting. Cancer drug resistance includes over-expression of drug efflux transporters, hypoxic environment, and defective apoptotic pathways. To overcome these discrepancies, a lot of nanomaterials (NMs), as emerging delivery system, have attracted attention for carrying and delivering therapeutics to the desired sites of interest. For improving the targeting potential of the anticancer cargos, NMs are optimized for their sizes, shapes, and surfaces for enhancing their circulation time and targeting efficiency. The NMs through encapsulation or conjugation of cargos with ligands may target them to the cancer site/s with enhanced therapeutic efficacy in a controlled release manner. Generally, NMs, as cancer therapy, are utilized to target cancer cells, tumor microenvironment, and immune system mainly through enhanced permeability and retention (EPR) effect¸ stimuli-responsive targeting or modifying their surfaces with targeting ligands such as transferin, integrins, sugar, folic acid and antibodies for the enhancement of tissue targeting recognition and their internalization. This review demonstrates mainly the biomedical applications of NMs as delivery vehicles or systems functionalized with ligands and therapeutic agents to target cancer cells / tissues passively, actively or physically with higher inhibitory therapeutic efficacies against cancer. Keywords: Cancer; Conventional chemotherapy; Nanomaterials; Biomedical applications; Therapeutic targeting; Inhibitory therapeutic efficacies
... Ferritin specifically targets on the tumor cells, which has been applied to tumor visualization and 395 targeted drug delivery(Fan et al., 2012;Fan et al., 2018). Our study shows that the block of ferritin 396 or iron can inhibit tumor effectively, which offers an explanation that ferritin targets on tumor is to 397 transport iron for maintaining the cell proliferation, highlighting a potential target for tumor 398Iron is an essential component for Fe-S clusters which participate in TCA and OxPhos, providing 400 energy required for self-renewal and proliferation of NBs. ...
Stem cell niche is critical for regulating the behavior of stem cells. Drosophila neural stem cells (Neuroblasts, NBs) are encased by glial niche cells closely, but it still remains unclear whether glial niche cells can regulate the self-renewal and differentiation of NBs. Here we show that ferritin produced by glia, cooperates with Zip13 to transport iron into NBs for the energy production, which is essential to the self-renewal and proliferation of NBs. The knockdown of glial ferritin encoding genes causes energy shortage in NBs, which leads to the low proliferation and premature differentiation of NBs. Moreover, the level of glial ferritin production is affected by the status of NBs, establishing a bicellular iron homeostasis. In this study, we demonstrate that glial cells are indispensable to maintain the self-renewal of NBs, unveiling a novel role of the NB glial niche during brain development.
... In particular, human heavy chain ferritin (HFn) has garnered attention as an effective drug delivery carrier for cancer therapy [10][11][12][13] . Through its specific binding to transferrin receptor 1 (TfR1), ferritin can specifically target cancer cells [14][15][16] . Ferritin, a conserved protein renowned for its iron binding and storage capabilities, possesses a self-assembling structure comprised of a hollow protein shell and this shell exhibits an outer diameter measuring 12 nm and an inner diameter measuring 8 nm [17][18][19][20] . ...
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.
An overview of the advancements in protein nanoparticles for immunotherapy across diseases include cancer and other diseases like influenza, pneumonia, and hepatitis.
Nanozymes, as an emerging class of enzyme mimics, have attracted much attention due to their adjustable catalytic activity, low cost, easy modification, and good stability. Researchers have made great efforts in developing and applying high-performance nanozymes. Recently, transition-metal-based nanozymes have been designed and widely developed because they possess unique photoelectric properties and high enzyme-like catalytic activities. To highlight these achievements and help researchers to understand the research status of transition-metal-based nanozymes, the development of transition-metal-based nanozymes from material characteristics to biological applications is summarized. Herein, we focus on introducing six categories of transition-metal-based nanozymes and highlight their progress in biomarker sensing and catalytic therapy for tumors. We hope that this review can guide the further development of transition-metal-based nanozymes and promote their practical applications in cancer diagnosis and treatment.
Leukemia is a widespread hematological malignancy characterized by an elevated white blood cell count in both the blood and the bone marrow. Despite notable advancements in leukemia intervention in the clinic, a large proportion of patients, especially acute leukemia patients, fail to achieve long‐term remission or complete remission following treatment. Therefore, leukemia therapy necessitates optimization to meet the treatment requirements. In recent years, a multitude of materials have undergone rigorous study to serve as delivery vectors or direct intervention agents to bolster the effectiveness of leukemia therapy. These materials include liposomes, protein‐based materials, polymeric materials, cell‐derived materials, and inorganic materials. They possess unique characteristics and are applied in a broad array of therapeutic modalities, including chemotherapy, gene therapy, immunotherapy, radiotherapy, hematopoietic stem cell transplantation, and other evolving treatments. Here, an overview of these materials is presented, describing their physicochemical properties, their role in leukemia treatment, and the challenges they face in the context of clinical translation. This review inspires researchers to further develop various materials that can be used to augment the efficacy of multiple therapeutic modalities for novel applications in leukemia treatment.
This paper explores the intricate interplay between inorganic nanoparticles and Earth's biochemical history, with a focus on their electron transfer properties. It reveals how iron oxide and sulfide nanoparticles, as examples of inorganic nanoparticles, exhibit oxidoreductase activity similar to proteins. Termed "life fossil oxidoreductases", these inorganic enzymes influence redox reactions, detoxification processes, and nutrient cycling in early Earth environments. By emphasizing the structural configuration of nanoparticles and their electron conformation, including oxygen defects and metal vacancies, especially electron hopping, the paper provides a foundation for understanding inorganic enzyme mechanisms. This approach, rooted in physics, underscores that life's origin and evolution are governed by electron transfer principles within the framework of chemical equilibrium. Today, these nanoparticles serve as vital biocatalysts in natural ecosystems, participating in critical reactions for ecosystem health. The research highlights their enduring impact on Earth's history, shaping ecosystems and interacting with protein metal centers through shared electron transfer dynamics, offering insights into early life processes and adaptations.
The effects of an increasingly elderly population are among the most far-reaching in 21st-century society. The growing healthcare expense is mainly attributable to the increased incidence of chronic illnesses that accompany longer life expectancies. Different ideas have been put up to explain aging, but it is widely accepted that oxidative damage to proteins, lipids, and nucleic acids contributes to the aging process. Increases in life expectancy in all contemporary industrialized cultures are accompanied by sharp increases in the prevalence of age-related diseases such as cardiovascular and neurological conditions, type 2 diabetes, osteoporosis, and cancer. Therefore, academic and public health authorities should prioritize the development of therapies to increase health span. Nanozyme (NZ)-like activity in nanomaterials has been identified as promising anti-aging nanomedicines. More than that, nanomaterials displaying catalytic activities have evolved as artificial enzymes with high structural stability, variable catalytic activity, and functional diversity for use in a wide range of biological settings, including those dealing with age-related disorders. Due to their inherent enzyme-mimicking qualities, enzymes have attracted significant interest in treating diseases associated with reactive oxygen species (ROS). The effects of NZs on aging and age-related disorders are summarized in this article. Finally, prospects and threats to enzyme research and use in aging and age-related disorders are offered.
Graphical Abstract
Comprehensive Summary
We have compiled eight promising strategies for enhancing the specificity and selectivity of nanozymes, as depicted in the comprehensive summary above. Enzymes exhibit intricate and sophisticated structures, including substrate channels and active sites, which can inform the design of nanozymes. Replication of these structural features and the application of facet engineering/doping techniques can significantly enhance the catalytic specificity of nanozymes. Alternatively, the use of Molecularly Imprinted Polymers (MIPs) to coat nanozymes represents an effective approach to impart substrate specificity. Furthermore, several straightforward stopgap strategies have been devised to improve nanozyme specificity for analytical applications, such as the integration of biorecognition elements and nanozyme sensor arrays through surface modification. image
Key Scientists
image
Iron oxide nanozymes (IONzymes) are a class of magnetic nanoparticles that mimic the enzymatic activity of natural enzymes. These particles have received significant attention in recent years due to their unique properties, such as high stability, tunable magnetic responsiveness, and ability to act as biocatalysts for various chemical reactions. In this review, we aim to provide an overview of the production methods of magnetic nanozymes, including chemical, physical, and biological synthesis. The structure and design of magnetic nanozymes are also discussed in detail, as well as their applications in various fields such as biomedicine and environmental science. The results of various studies and the latest advances in the field of magnetic nanozymes are also discussed. This review provides valuable insights into the current state of magnetic nanozymes and highlights their potential for further development and application in various fields.
Well‐defined nanostructures are crucial for precisely understanding nano‐bio interactions. However, nanoparticles (NPs) fabricated through conventional synthesis approaches often lack poor controllability and reproducibility. Herein, a synthetic biology‐based strategy is introduced to fabricate uniformly reproducible protein‐based NPs, achieving precise control over heterogeneous components of the NPs. Specifically, a ferritin assembly toolbox system is developed that enables intracellular assembly of ferritin subunits/variants in Escherichia coli. Using this strategy, a proof‐of‐concept study is provided to explore the interplay between ligand density of NPs and their tumor targets/penetration. Various ferritin hybrid nanocages (FHn) containing human ferritin heavy chains (FH) and light chains are accurately assembled, leveraging their intrinsic binding with tumor cells and prolonged circulation time in blood, respectively. Further studies reveal that tumor cell uptake is FH density‐dependent through active binding with transferrin receptor 1, whereas in vivo tumor accumulation and tissue penetration are found to be correlated to heterogeneous assembly of FHn and vascular permeability of tumors. Densities of 3.7 FH/100 nm² on the nanoparticle surface exhibit the highest degree of tumor accumulation and penetration, particularly in tumors with high permeability compared to those with low permeability. This study underscores the significance of nanoparticle heterogeneity in determining particle fate in biological systems.
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.
Iron oxide nanoparticles with intrinsic enzymatic properties are considered as iron oxide nanozymes (IONzymes). IONzymes are bridging the gap between nanotechnology and biomedicine. Their catalytic actions mimic those of natural enzymes such as peroxidase and catalase. IONzymes are synthesized chemically and are less expensive than the preparation of natural enzymes. They are stable over a wide range of pH and temperature, an advantage over natural enzymes. In addition to IONzyme’s enzymatic properties, their magnetic properties provide opportunities for developing of bioseparation assays, imaging tools, targeted drug delivery and hyperthermia therapy in the field of biomedicine.
We investigated the relevance of encapsulation in H-ferritin nanocages (HFn) in determining an improved tumor-targeted delivery of indocyanine green (ICG). Since from previous experiments, the administration of HFn loaded with ICG (HFn-ICG) resulted in an increased fluorescence signal of ICG, our aim was to uncover if the nanoformulation could have a major role in driving a specific targeting of the dye to the tumor or rather a protective action on ICG’s fluorescence. Here, we took advantage of a combined analysis involving ultrahigh performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) on murine tissue homogenates matched with fluorescence intensities analysis detected by ex vivo optical imaging. The quantification of ICG content performed on different organs over time combined with the fluorescent signal detection confirmed the superior delivery of ICG thanks to the nanoformulation. Our results showed that HFn-ICG drives a real accumulation at the tumor instead of only having a role in the preservation of ICG’s fluorescence, further supporting its use as a delivery system of ICG for fluorescence-guided surgery applications in oncology.
Hydrogels are prevailing drug delivery depots to improve antitumor efficacy and reduce systemic toxicity. However, the application of conventional free drug‐loaded hydrogel is hindered by poor drug penetration in solid tumors. Here, an injectable ferritin‐based nanocomposite hydrogel is constructed to facilitate tumor penetration and improve cancer chemoimmunotherapy. Specifically, doxorubicin‐loaded human ferritin (Dox@HFn) and oxidized dextran (Dex‐CHO) are used to construct the injectable hydrogel (Dox@HFn Gel) through the formation of pH‐sensitive Schiff‐base bonds. After peritumoral injection, the Dox@HFn Gel is retained locally for up to three weeks, and released intact Dox@HFn gradually, which can not only facilitate tumor penetration through active transcytosis but also induce immunogenic cell death (ICD) to tumor cells to generate an antitumor immune response. Combining with anti‐programmed death‐1 antibody (αPD‐1), Dox@HFn Gel induces remarkable regression of orthotopic 4T1 breast tumors, further elicits a strong systemic anti‐tumor immune response to effectively suppress tumor recurrence and lung metastasis of 4T1 tumors after surgical resection. Besides, the combination of Dox@HFn GelL with anti‐CD47 antibody (αCD47) inhibits postsurgical tumor recurrence of aggressive orthotopic glioblastoma tumor model and significantly extends mice survival. This work sheds light on the construction of local hydrogels to potentiate antitumor immune response for improved cancer therapy.
Nowadays, environmental contamination is diverse and complicated, and the environment is polluted by multiple harmful chemicals from anthropogenic sources, necessitating the development of efficient and low-cost technologies for monitoring them. In this paper, the hydrothermal technique has been used to synthesize naked Fe3O4, PEG-coated Fe3O4, and CTAB-coated Fe3O4 magnetic nanoparticles (MNPs). X-ray diffraction studies showed that the crystallite sizes of the naked Fe3O4, PEG-coated Fe3O4, and CTAB-coated Fe3O4 NPs were 47.23, 39.23, and 31.70 nm, respectively. All the products had a spherical shape with a hollow structure, according to the FESEM analysis. The VSM revealed that the saturation magnetization of the samples ranged from 82.7 to 77.5 emu/g. By employing unmodified and modified magnetite nanozyme, we present a simple and high-performance sensor for colorimetric detection of phenol. The samples showed different catalytic activity in enhancing the reaction of 4-aminoantipyrine, phenolic species, and H2O2, resulting in a spectacular color shift to signal the target level. This ability of the products was used for the detection of phenol in concentrations ranging from 0.25 μM to 1.2 mM.
Drug delivery in human subjects has been the most difficult task since the ancient time of the medical sector. An ideal drug delivery system is, one that minimizes the adverse effects and maximizes the desired effects of the drug candidate. Various drug delivery systems have been developed that may have some kind of advantages and disadvantages, among them targeted drug delivery system is more preferable and convenient which may employ various nanoparticles or other materials for the drug delivery at the specified site of action. In this, the authors elaborately and comprehensively explained the role of recent carbon nanotubes (CNTs) in targeted drug delivery systems (specifically for targeting cancerous cells). The authors also described the methods of preparation of CNTs, characterization techniques for CNTs, cellular penetration of, CNTs, and the associated toxicities with CNTs. Carbon nanotubes are preferable to other nanoparticles because they are more electrically, mechanically, and organically stable than others, they can carry more amount of drug in comparison to other nanoparticles and their functionalization property makes them more attractive as a carrier molecule for targeting any root cause of the disease.
Despite the tremendous progress in cancer treatment in recent decades, cancers often become resistant due to multiple mechanisms, such as intrinsic or acquired multidrug resistance, which leads to unsatisfactory treatment effects or accompanying metastasis and recurrence, ultimately to treatment failure. With a deeper understanding of the molecular mechanisms of tumors, researchers have realized that treatment designs targeting tumor resistance mechanisms would be a promising strategy to break the therapeutic deadlock. Nanodelivery systems have excellent physicochemical properties, including highly efficient tissue‐specific delivery, substantial specific surface area, and controllable surface chemistry, which endow nanodelivery systems with capabilities such as precise targeting, deep penetration, responsive drug release, multidrug codelivery, and multimodal synergy, which are currently widely used in biomedical researches and bring a new dawn for overcoming cancer resistance. Based on the mechanisms of tumor therapeutic resistance, this review summarizes the research progress of nanodelivery systems for overcoming tumor resistance to improve therapeutic efficacy in recent years and offers prospects and challenges of the application of nanodelivery systems for overcoming cancer resistance.
The success of arsenic in degrading PML‐RARα oncoprotein illustrates the great anti‐leukemia value of inorganics. Inspired by this, the therapeutic effect of inorganic selenium on t(8; 21) leukemia is studied, which has shown promising anti‐cancer effects on solid tumors. A leukemia‐targeting selenium nanomedicine is rationally built with bioengineered protein nanocage and is demonstrated to be an effective epigenetic drug for inducing the differentiation of t(8;21) leukemia. The selenium drug significantly induces the differentiation of t(8;21) leukemia cells into more mature myeloid cells. Mechanistic analysis shows that the selenium is metabolized into bioactive forms in cells, which drives the degradation of the AML1‐ETO oncoprotein by inhibiting histone deacetylases activity, resulting in the regulation of AML1‐ETO target genes. The regulation results in a significant increase in the expression levels of myeloid differentiation transcription factors PU.1 and C/EBPα, and a significant decrease in the expression level of C‐KIT protein, a member of the type III receptor tyrosine kinase family. This study demonstrates that this protein‐nanocaged selenium is a potential therapeutic drug against t(8;21) leukemia through epigenetic regulation.
Co²⁺‐ZIF‐67 metal‐organic framework nanoparticles (NMOFs), act as nanozymes catalyzing diverse processes, including peroxidase, oxidase, and catalase activities. Peroxidase activities are reflected by the nanozyme‐catalyzed H2O2 oxidation of dopamine to aminochrome, the H2O2 oxidation of NADH to NAD⁺, the H2O2‐catalyzed generation of chemiluminescence through oxidation of luminol, and the H2O2‐mediated oxidation of aniline to polyaniline. Oxidase activities of the nanozyme are demonstrated by the Co²⁺‐ZIF‐67 NMOFs catalyzed aerobic oxidation of dopamine to aminochrome and of NADH to NAD⁺, and catalase activities of the nanozyme are reflected by the catalytic decomposition of H2O2. Moreover, the Co²⁺‐ZIF‐67 catalyzed oxidation of aniline to polyaniline (PAN) by H2O2 is accompanied by the coating of NMOFs with PAN to yield a Co²⁺‐ZIF‐67/PAN hybrid material. Coating the particles in the presence of guest substrates leads to molecular imprinted PAN matrices revealing enhanced ZIF‐67 catalyzed transformations as compared to non‐imprinted PAN matrices. This is demonstrated by imprinting of 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid), ABTS²⁻, in the PAN coating and the 3.3‐fold enhanced catalyzed oxidation of ABTS²⁻ by H2O2, as compared to the non‐imprinted PAN hybrid composite. Moreover, imprinting of L‐/D‐DOPA in the PAN coating of Co²⁺‐ZIF‐67 NMOFs leads to chiro‐selective H2O2‐guided oxidation of L‐/D‐DOPA to dopachrome by the NMOFs/PAN composite.
Glucose oxidase-loaded ZIF-90 metal-organic framework nanoparticles conjugated to hemin-G-quadruplexes act as functional bioreactor hybrids operating transient dissipative biocatalytic cascaded transformations consisting of the glucose-driven H2O2-mediated oxidation of Amplex-Red to resorufin or the glucose-driven generation of chemiluminescence by the H2O2-mediated oxidation of luminol. One system involves the fueled activation of a reaction module leading to the temporal formation and depletion of the bioreactor conjugate operating the nickase-guided transient biocatalytic cascades. The second system demonstrates the fueled activation of a reaction module yielding a bioreactor conjugate operating the exonuclease III-dictated transient operation of the two biocatalytic cascades. The temporal operations of the bioreactor circuits are accompanied by kinetic models and computational simulations enabling us to predict the dynamic behavior of the systems subjected to different auxiliary conditions.
Regenerable nanozymes with high catalytic stability and sustainability are promising substitutes for naturally-occurring enzymes but are limited by insufficient and non-selective catalytic activities. Herein, we developed single-atom nanozymes of RhN 4 , VN 4 , and Fe-Cu-N 6 with catalytic activities surpassing natural enzymes. Notably, Rh/VN 4 preferably forms an Rh/V-ON 4 active center to decrease reaction energy barriers and mediates a "two-sided oxygen-linked" reaction path, showing 4 and 5-fold higher affinities in peroxidase-like activity than the FeN 4 and natural horseradish peroxidase. Furthermore, RhN 4 presents a 20-fold improved affinity in the catalase-like activity compared to the natural catalase; Fe-Cu-N 6 displays selectivity towards the superoxide dismutase-like activity; VN 4 favors a 7-fold higher glutathione peroxidase-like activity than the natural glutathione peroxidase. Bioactive sutures with Rh/VN 4 show recyclable catalytic features without apparent decay in 1 month and accelerate the scalp healing from brain trauma by promoting the vascular endothelial growth factor, regulating the immune cells like mac-rophages, and diminishing inflammation. Artificial enzyme engineering with sustainabilities emerges as a versatile methodology for creating biocatalysts 1-6. The intrinsic feature of continuous electron transfers allows artificial enzymes to maintain high catalytic stability and arbitrarily tailor their characteristics 7-12. The superior catalytic activity over the naturally-occurring enzymes is one of the major challenges existing in designing artificial enzymes 13-16. A feasible strategy is to construct an artificial active center by imitating the biologically active center of enzymes or proteins 2,17-22. As a fast growing category of artificial enzymes, the emerging nanozyme can actively tailor biocatalytic activities and selectivity via its flexible atomic structures and molecular engineering 23-26. State-of-art nano-zymes with an M-N 4 center inspired by cytochrome P450 have superior reaction rates and high substrate affinities close to the natural horseradish peroxidase (HRP) 27-29. Expanded Fe-N 5 and Fe-N 3-P were reported to have 17-and 2-fold higher catalytic efficiencies than Fe-N 4 14,30 , indicating their great potential in boosting bioactivities. However , the development of nanozymes with properties outperforming natural enzymes remains unfulfilled and highly challenging. Other unresolved and high-profile questions are the underlying reaction mechanisms and detailed atomic coordination structures
While the primary use of protein crystals has historically been in crystallographic structure determination, they have recently emerged as promising materials with many advantageous properties such as high porosity, biocompatibility, stability, structural and functional versatility, and genetic/chemical tailorability. Here, we report that the utility of protein crystals as functional materials can be further augmented through their spatial patterning and control of their morphologies. To this end, we took advantage of the chemically and kinetically controllable nature of ferritin self-assembly and constructed core-shell crystals with chemically distinct domains, tunable structural patterns, and morphologies. The spatial organization within ferritin crystals enabled the generation of patterned, multi-enzyme frameworks with cooperative catalytic behavior. We further exploited the differential growth kinetics of ferritin crystal facets to assemble Janus-type architectures with an anisotropic arrangement of chemically distinct domains. These examples represent a step toward using protein crystals as reaction vessels for complex multi-step reactions and broadening their utility as functional, solid-state materials. Our results demonstrate that morphology control and spatial patterning, which are key concepts in materials science and nanotechnology, can also be applied for engineering protein crystals.
Early noninvasive detection and characterization of solid tumors and their supporting neovasculature is a fundamental prerequisite for effec- tive therapeutic intervention, particularly antiangiogenic treatment regi- mens. Emerging molecular imaging techniques now allow recognition of early biochemical, physiological, and anatomical changes before manifes- tation of gross pathological changes. Although new tumor, vascular, ex- tracellular matrix, and lymphatic biomarkers continue to be discovered, the 3-integrin remains an attractive biochemical epitope that is highly expressed on activated neovascular endothelial cells and essentially absent on mature quiescent cells. In this study, we report the first in vivo use of a magnetic resonance (MR) molecular imaging nanoparticle to sensitively detect and spatially characterize neovascularity induced by implantation of the rabbit Vx-2 tumor using a common clinical field strength (1.5T). New Zealand White rabbits (2 kg) 12 days after implantation of fresh Vx-2 tumors (2 2 2m m 3) were randomized into one of three treatment groups: (a) 3-targeted, paramagnetic formulation; (b) nontargeted, paramagnetic formulation; and (c) 3-targeted nonparamagnetic nano- particles followed by (2 h) the 3-targeted, paramagnetic formulation to competitively block magnetic resonance imaging (MRI) signal enhance- ment. After i.v. systemic injection (0.5 ml of nanoparticles/kg), dynamic T1-weighted MRI was used to spatially and temporally determine nano- particle deposition in the tumor and adjacent tissues, including skeletal muscle. At 2-h postinjection, 3-targeted paramagnetic nanoparticles increased MRI signal by 126% in asymmetrically distributed regions primarily in the periphery of the tumor. Similar increases in MR contrast were also observed within the walls of some vessels proximate to the tumor. Despite their relatively large size, nanoparticles penetrated into the leaky tumor neovasculature but did not appreciably migrate into the interstitium, leading to a 56% increase in MR signal at 2 h. Pretargeting of the 3-integrin with nonparamagnetic nanoparticles competitively blocked the specific binding of 3-targeted paramagnetic nanoparticles, decreasing the MR signal enhancement (50%) to a level attributable to local extravasation. The MR signal of adjacent hindlimb muscle or con- tralateral control tissues was unchanged by either the 3-targeted or control paramagnetic agents. Immunohistochemistry of 3-integrin corroborated the extent and asymmetric distribution of neovascularity observed by MRI. These studies demonstrate the potential of this targeted molecular imaging agent to detect and characterize (both biochemically and morphologically) early angiogenesis induced by minute solid tumors with a clinical 1.5 Tesla MRI scanner, facilitating the localization of nascent cancers or metastases, as well as providing tools to phenotypically categorize and segment patient populations for therapy and to longitudi- nally follow the effectiveness of antitumor treatment regimens.
The spread of cancer cells between organs, a process known as metastasis, is the cause of most cancer deaths1, 2. Detecting circulating tumour cells—a common marker for the development of metastasis3, 4—is difficult because ex vivo methods are not sensitive enough owing to limited blood sample volume and in vivo diagnosis is time-consuming as large volumes of blood must be analysed5, 6, 7. Here, we show a way to magnetically capture circulating tumour cells in the bloodstream of mice followed by rapid photoacoustic detection. Magnetic nanoparticles, which were functionalized to target a receptor commonly found in breast cancer cells, bound and captured circulating tumour cells under a magnet. To improve detection sensitivity and specificity, gold-plated carbon nanotubes conjugated with folic acid were used as a second contrast agent for photoacoustic imaging. By integrating in vivo multiplex targeting, magnetic enrichment, signal amplification and multicolour recognition, our approach allows circulating tumour cells to be concentrated from a large volume of blood in the vessels of tumour-bearing mice, and this could have potential for the early diagnosis of cancer and the prevention of metastasis in humans.
Acute leukemia is a hematopoietic malignancy for which the accurate measurement of minimal residual disease is critical to determining prognosis and treatment. Although bone marrow aspiration and light microscopy remain the current standard of care for detecting residual disease, these approaches cannot reliably discriminate less than 5% lymphoblast cells. To improve the detection of leukemia cells in the marrow, we developed a novel apparatus that utilizes antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPION) and directed against the acute leukemia antigen CD34, coupled with a "magnetic needle" biopsy. Leukemia cell lines expressing high or minimal CD34 were incubated with anti-CD34-conjugated SPIONs. Three separate approaches including microscopy, superconducting quantum interference device magnetometry, and in vitro magnetic needle extraction were then used to assess cell sampling. We found that CD34-conjugated nanoparticles preferentially bind high CD34-expressing cell lines. Furthermore, the magnetic needle enabled identification of both cell line and patient leukemia cells diluted into normal blood at concentrations below those normally found in remission marrow samples. Finally, the magnetic needle enhanced the percentage of lymphoblasts detectable by light microscopy by 10-fold in samples of fresh bone marrow aspirate approximating minimal residual disease. These data suggest that bone marrow biopsy using antigen-targeted magnetic nanoparticles and a magnetic needle for the evaluation of minimal residual disease in CD34-positive acute leukemias can significantly enhance sensitivity compared with the current standard of care. [Cancer Res 2009;69(21):8310-6]
A highly efficient process using iron oxide magnetic nanoparticles (IO)-based immunomagnetic separation of tumor cells from fresh whole blood has been developed. The process involved polymer coated 30 nm IO that was modified with antibodies (Ab) against human epithelial growth factor receptor 2 (anti-HER2 or anti-HER2/neu) forming IO-Ab. HER2 is a cell membrane protein that is overexpressed in several types of human cancer cells. Using a HER2/neu overexpressing human breast cancer cell line, SK-BR3, as a model cell, the IO-Ab was used to separate 73.6% (with a maximum capture of 84%) of SK-BR3 cells that were spiked in 1 mL of fresh human whole blood. The IO-Ab preferentially bound to SK-BR3 cells over normal cells found in blood due to the high level of HER2/neu receptor on the cancer cells unlike the normal cell surfaces. The results showed that the nanosized magnetic nanoparticles exhibited an enrichment factor (cancer cells over normal cells) of 1:10,000,000 in a magnetic field (with gradient of 100 T/m) through the binding of IO-Ab on the cell surface that resulted in the preferential capture of the cancer cells. This research holds promise for efficient separation of circulating cancer cells in fresh whole blood.
Nanoparticles containing magnetic materials, such as magnetite (Fe3O4), are particularly useful for imaging and separation techniques. As these nanoparticles are generally considered to be biologically and chemically inert, they are typically coated with metal catalysts, antibodies or enzymes to increase their functionality as separation agents. Here, we report that magnetite nanoparticles in fact possess an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, which are widely used to oxidize organic substrates in the treatment of wastewater or as detection tools. Based on this finding, we have developed a novel immunoassay in which antibody-modified magnetite nanoparticles provide three functions: capture, separation and detection. The stability, ease of production and versatility of these nanoparticles makes them a powerful tool for a wide range of potential applications in medicine, biotechnology and environmental chemistry.
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.
Nanoparticle technologies are significantly impacting the development of both therapeutic and diagnostic agents. At the intersection between treatment and diagnosis, interest has grown in combining both paradigms into clinically effective formulations. This concept, recently coined as theranostics, is highly relevant to agents that target molecular biomarkers of disease and is expected to contribute to personalized medicine. Here we review state-of-the-art nanoparticles from a therapeutic and a diagnostic perspective and discuss challenges in bringing these fields together. Major classes of nanoparticles include, drug conjugates and complexes, dendrimers, vesicles, micelles, core-shell particles, microbubbles, and carbon nanotubes. Most of these formulations have been described as carriers of either drugs or contrast agents. To observe these formulations and their interactions with disease, a variety of contrast agents have been used, including optically active small molecules, metals and metal oxides, ultrasonic contrast agents, and radionuclides. The opportunity to rapidly assess and adjust treatment to the needs of the individual offers potential advantages that will spur the development of theranostic agents.
The emerging field of bionanotechnology aims at revolutionizing biomedical research and clinical practice via introduction of nanoparticle-based tools, expanding capabilities of existing investigative, diagnostic, and therapeutic techniques as well as creating novel instruments and approaches for addressing challenges faced by medicine. Quantum dots (QDs), semiconductor nanoparticles with unique photo-physical properties, have become one of the dominant classes of imaging probes as well as universal platforms for engineering of multifunctional nanodevices. Possessing versatile surface chemistry and superior optical features, QDs have found initial use in a variety of in vitro and in vivo applications. However, careful engineering of QD probes guided by application-specific design criteria is becoming increasingly important for successful transition of this technology from proof-of-concept studies towards real-life clinical applications. This review outlines the major design principles and criteria, from general ones to application-specific, governing the engineering of novel QD probes satisfying the increasing demands and requirements of nanomedicine and discusses the future directions of QD-focused bionanotechnology research (critical review, 201 references).
Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules.
Akt/protein kinase B signaling pathway has been implicated in tumorigenesis and progression. Previous studies showed the predictive potential of p-Akt-1, but total Akt-1 could provide more reliable information. We used image deconvolution, nanotechnology (quantum dots), and image analysis to improve Akt-1 quantification.
This tissue microarray study included 840 radical prostatectomy cases. Slides were incubated with primary antibody against nonphosphorylated Akt-1 (Akt-1) followed by biotinylated secondary antibody and then by Qdot655 streptavidin conjugate. Slides were imaged under fluorescence microscopy and spectral deconvolution (Nuance) and quantified using plug-in image analysis software. Average intensity of Akt-1 signal was measured and subject to statistical analysis. Multivariate analysis (Cox regression) was applied to assess the prognostic value of Akt-1 for biochemical recurrence and prostate cancer-specific death. Akt-1 expression was also examined for correlations with Ki-67 index and apoptotic index in our database.
Akt-1 was inversely correlated with apoptotic index (rho = -0.203; P = 0.004) but not with Ki-67 index. The correlation between Akt and p-Akt is significant but weak (P = 0.0496; R(2) = 0.118). On multivariate analysis Akt-1 was independently predictive of biochemical recurrence [hazard ratio, 2.863 (95% confidence interval, 1.127-7.271); P = 0.0270]. Akt-1 level is also predictive of prostate cancer-specific death (P = 0.0376).
High levels of Akt-1, assessed by quantum dots, deconvolution imaging, and image analysis, are associated with a higher risk of biochemical recurrence and prostate cancer-specific death.
We describe the development of multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer and linking this amphiphilic polymer to tumor-targeting ligands and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes accumulate at tumors both by the enhanced permeability and retention of tumor sites and by antibody binding to cancer-specific cell surface biomarkers. Using both subcutaneous injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes, we have achieved sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. We have also integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of molecular targets in vivo.
Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades
from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization,
and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use
include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes
in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the
study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation
of cell trafficking, tumor targeting, and diagnostics.
Nanomaterials with precise biological functions have considerable potential for use in biomedical applications. Here we investigate whether multivalent attachment of small molecules can increase specific binding affinity and reveal new biological properties of such nanomaterials. We describe the parallel synthesis of a library comprising 146 nanoparticles decorated with different synthetic small molecules. Using fluorescent magnetic nanoparticles, we rapidly screened the library against different cell lines and discovered a series of nanoparticles with high specificity for endothelial cells, activated human macrophages or pancreatic cancer cells. Hits from the last-mentioned screen were shown to target pancreatic cancer in vivo. The method and described materials could facilitate development of functional nanomaterials for applications such as differentiating cell lines, detecting distinct cellular states and targeting specific cell types.
Homogeneous magnetic nanoparticles can be synthesised within the constrained volume architecture of Listeria innocua (see Figure). This ferritin‐like protein cage provides a limited reaction environment for the synthesis of 5 nm diameter ferrimagnetic iron oxide maghemite nanoparticles. The results suggest a new general synthesis route for nanomaterials, utilizing the inherent host–guest properties of protein cage architectures.
Both inorganic nanoparticles and polymers have become ubiquitous components of nanostructured systems. The development of an understanding of the interactions between these components will be of crucial importance in the further elaboration of these systems. This review touches upon two aspects of control over surface chemistry in inorganic nanoparticles with polymer ligands: (1) the effect of polymer ligands on accessibility of the inorganic nanoparticle surface to other species, such as is necessary in catalysis; and (2) the design of polymer ligands that can be modified to enable compatibility of the entire polymer‐coated nanoparticle with a broad variety of other materials.
Nanotechnology has had tremendous impact on medical science and has resulted in phenomenal progress in the field of drug delivery and diagnostics. A wide spectrum of novel nanomaterials including polymeric particles, liposomes, quantum dots, and iron oxide particles have been developed for applications in therapeutic delivery and diagnostics. This has resulted in control over the rate and period of delivery and targeting of drugs to specific organs in the human body. This feature article focuses on the delivery of drugs using polymeric particles. The size, choice of polymer, surface chemistry, shape, and mechanical properties of the particles are parameters that critically affect particle function. Numerous biomaterials and fabrication techniques have been developed in the last decade that focus on novel design parameters, such as shape and mechanical properties and the interplay of these parameters with the size and surface chemistry of particles. Recent advances with particular focus on the importance of particle shape are highlighted, and the challenges that are yet to be fulfilled are underscored.
The protein cage constrained synthesis of ferrimagnetic iron oxide nanoparticles was presented. The ferritin-like protein (FLP) from the bacteria Listeria innocua was cloned into an inducible E. coli heterologous expression system and was analyzed by size exclusion chromatography (SEC), transmission electron microscopy (TEM) and dynamic light scattering (DLS). The Fe mineralization in the FLP cage under the non-physiological conditions of elevated temperature, pH and controlled oxidation was also investigated.
1] Studying the magnetic properties of ultrafine nanometer‐scale ferrimagnetic particles (<10 nm) is vital to our understanding of superparamagnetism and its applications to environmental magnetism, biogeomagnetism, iron biomineralization, and biomedical technology. However, magnetic properties of the ultrafine nanometer‐sized ferrimagnetic grains are very poorly constrained because of ambiguities caused by particle magnetostatic interactions and unknown size distributions. To resolve these problems, we synthesized magnetoferritins using the recombinant human H chain ferritin (HFn). These ferrimagnetic HFn were further purified through size exclusion chromatography to obtain monodispersed ferrimagnetic HFn. Transmission electron microscopy revealed that the purified ferrimagnetic HFn are monodispersed and each consists of an iron oxide core (magnetite or maghemite) with an average core diameter of 3.9 ± 1.1 nm imbedded in an intact protein shell. The R value of the Wohlfarth‐Cisowski test measured at 5 K is 0.5, indicating no magnetostatic interactions. The saturation isothermal remanent magnetization acquired at 5 K decreased rapidly with increasing temperature with a median unblocking temperature of 8.2 K. The preexponential frequency factor f 0 determined by AC susceptibility is (9.2 ± 7.9) × 10 10 Hz. The extrapolated M rs /M s and B cr /B c at 0 K are 0.5 and 1.12, respectively, suggesting that the ferrimagnetic HFn cores are dominated by uniaxial anisotropy. The calculated effective magnetic anisotropy energy constant K eff = 1.2 × 10 5 J/m 3 , which is larger than previously reported values for bulk magnetite and/or maghemite or magnetoferritin and is attributed to the effect of surface anisotropy. These data provide useful insights into superparamagnetism as well as biomineralization of ultrafine ferrimagnetic particles.
Nanoscale therapeutic interventions are increasingly important elements in the portfolio of cancer therapeutics. The efficacy of nanotherapeutics is dictated, in part, by the access they have to tumors via the leaky tumor vasculature. Yet, the extent of tumor vessel leakiness in individual tumors varies widely resulting in a correspondingly wide tumor dosing and resulting range of responses to therapy. Here we report the design of a multifunctional nanocarrier that simultaneously encapsulates a chemotherapeutic and a contrast agent which enables a personalized nanotherapeutic approach for breast cancer therapy by permitting tracking of the nanocarrier distribution by mammography, a widely used imaging modality. Following systemic administration in a rat breast tumor model, imaging demonstrated a wide range of intratumoral deposition of the nanocarriers, indicating variable tumor vessel leakiness. Notably, specific tumors that exhibited high uptake of the nanocarrier as visualized by imaging were precisely the animals that responded best to the treatment as quantified by low tumor growth and prolonged survival.
Subcellular destiny of targeted nanoparticles in cancer cells within living organisms is still an open matter of debate. By in vivo and ex vivo experiments on tumor-bearing mice treated with antibody-engineered magnetofluorescent nanocrystals, in which we combined fluorescence imaging, magnetic relaxation, and trasmission electron microscopy approaches, we provide evidence that nanoparticles are effectively delivered to the tumor by active targeting. These nanocrystals were demonstrated to enable contrast enhancement of the tumor in magnetic resonance imaging. In addition, we were able to discriminate between the fate of the organic corona and the metallic core upon cell internalization. Accurate immunohistochemical analysis confirmed that hybrid nanoparticle endocytosis is mediated by the complex formation with HER2 receptor, leading to a substantial downregulation of HER2 protein expression on the cell surface. These results provide a direct insight into the pathway of internalization and degradation of targeted hybrid nanoparticles in cancer cells in vivo and suggest a potential application of this immunotheranostic nanoagent in neoadjuvant therapy of cancer.
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.
Engineering compact imaging probes with highly integrated modalities is a key focus in bionanotechnology and will have profound impact on molecular diagnostics, imaging and therapeutics. However, combining multiple components on a nanometre scale to create new imaging modalities unavailable from individual components has proven to be challenging. In this paper, we demonstrate iron oxide and gold-coupled core-shell nanoparticles (NPs) with well-defined structural characteristics (for example, size, shell thickness and core-shell separation) and physical properties (for example, electronic, magnetic, optical, thermal and acoustic). The resulting multifunctional nanoprobes not only offer contrast for electron microscopy, magnetic resonance imaging and scattering-based imaging but, more importantly, enable a new imaging mode, magnetomotive photoacoustic imaging, with remarkable contrast enhancement compared with photoacoustic images using conventional NP contrast agents.
One of the major limitations impeding the sensitivity and specificity of biomarker targeted nanoparticles is non-specific binding by biomolecules and uptake by the reticuloendothelial system (RES). We report the development of an antibiofouling polysiloxane containing amphiphilic diblock copolymer, poly(ethylene oxide)-block-poly(gamma-methacryloxypropyl trimethoxysilane) (PEO-b-PgammaMPS), for coating and functionalizing high quality hydrophobic nanocrystals such as iron oxide nanoparticles and quantum dots. These PEO-b-PgammaMPS-coated nanocrystals were colloidally stable in biological medium and showed low non-specific binding by macromolecules after incubation with 100% fetal bovine serum. Both in vitro experiments with macrophages and in vivo biodistribution studies in mice revealed that PEO-b-PgammaMPS copolymer-coated nanocrystals have an antibiofouling effect that reduces non-specific cell and RES uptake. Surface functionalization with amine groups was accomplished through co-crosslinking the polysiloxane coating layer and (3-Aminopropyl)trimethoxysilane in aqueous solution. Tumor integrin alpha(v)beta(3) targeting peptide cyclo-RGD ligands were conjugated on the nanoparticles through a heterobifunctional linker. The resulting integrin alpha(v)beta(3) targeting nanoparticle conjugates showed improved cancer cell targeting with a stronger affinity to U87MG glioma cells, which have a high expression of alpha(v)beta(3) integrins, but minimal binding to MCF-7 breast cancer cells with low expression of alpha(v)beta(3) integrins.
Self-assembled cage structures of nanometre dimensions can be used as constrained environments for the preparation of nanostructured materials, and the encapsulation of guest molecules, with potential applications in drug delivery and catalysis. In synthetic systems the number of subunits contributing to cage structures is typically rather small,. But the protein coats of viruses (virions) commonly comprise hundreds of subunits that self-assemble into a cage for transporting viral nucleic acids. Many virions, moreover, can undergo reversible structural changes that open or close gated pores to allow switchable access to their interior. Here we show that such a virion - that of the cowpea chlorotic mottle virus - can be used as a host for the synthesis of materials. We report the mineralization of two polyoxometalate species (paratungstate and decavanadate) and the encapsulation of an anionic polymer inside this virion, controlled by pH-dependent gating of the virion's pores. The diversity in size and shape of such virus particles make this a versatile strategy for materials synthesis and molecular entrapment.
The iron storage protein ferritin consists of a spherical polypeptide shell (apoferritin) surrounding a 6-nanometer inorganic core of the hydrated iron oxide ferrihydrite (5Fe2O3.9H2O). Previous studies have shown that the in vitro reconstitution of apoferritin yields mineral cores essentially identical to those of the native proteins. A magnetic mineral was synthesized within the nanodimensional cavity of horse spleen ferritin by the use of controlled reconstitution conditions. Transmission electron microscopy and electron diffraction analysis indicate that the entrapped mineral particles are discrete 6-nanometer spherical single crystals of the ferrimagnetic iron oxide magnetite (Fe3O4). The resulting magnetic protein, "magnetoferritin," could have uses in biomedical imaging, cell labeling, and separation procedures.
Using a panel of five monoclonal anti-transferrin receptor antibodies, we investigated the transferrin receptor expression in 34 human hepatocellular carcinomas of Belgian (n = 6), Italian (n = 7) and South African (n = 21) origin. For comparison the tumours were also stained with the monoclonal antibody BK 19.9, recognizing an antigen biochemically similar to the transferrin receptor, and with a monoclonal antibody against the epidermal growth factor receptor. Hepatocellular carcinomas express large amounts of transferrin receptors as demonstrated by the intense transferrin receptor immunostaining in 33/34 cases. Differences in staining pattern between and within the tumours were not related to the degree of tumour differentiation, nor to the origin or race of the patient. In 15 cases which included non-tumoural tissue, the tumour was more intensely stained than the surrounding liver parenchyma. The BK 19.9 immunoreactivity was generally weaker and mainly involved stromal cells, except in three cases where an intense staining of the tumour cells was seen. The epidermal growth factor receptor staining was also weaker and only in four cases was the immunoreactivity of the tumour stronger than the surrounding parenchyma. Demonstration of the transferrin receptor may be useful for the detection of malignant foci in liver biopsies. This may be of particular interest in the histological investigation of minute hepatocellular carcinomas.
The expression of transferrin receptor by normal, pregnant and benign hyperplastic breast lesions and by breast carcinomas has been investigated immunohistochemically using two monoclonal antibodies directed against the receptor. Unlike a previous immunohistological study in which staining was confined to imlignant breast, transferrin receptor has been detected in pregnant breast and in benign lesions as well as in all carcinomas examined. The latter showed variable reactivity but with staining of most cells in 70 percent of cases.
Although the expression of transfcrrin reccptor in non‐malignant conditions may be related to cell proliferation, as has been suggested from studies of activated cells, the extent of reactivity of carcinomas has shown no correlation with tumour characteristics such as differentiation and local tumour spread. It is therefore suggested that the immunologically active transferrin receptor of breast carcinomas may have significance other than that relating to proliferation. The finding that with some carcinomas differences in staining occurred between the two antibodies is a further illustration of the complexities of the nature of Itransfcrrin receptor.
Ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated by high resolution transmission electron microscopy, electron diffraction and chemical analysis. Hemosiderin particles isolated from thalassemic spleens also have been studied. The results show that there is a marked difference in structure and composition of the biomineral phases. Human ferritin and hemosiderin particles are single domain crystals of hydrated iron (III) oxide (ferrihydrite). Lattice fringes were low in contrast and often discontinuous within the central regions of the core. Heat treatment of human ferritins results in a 5 A shrinkage in particle size and an increase in the single crystalline nature of the core. In contrast, lattice images and electron diffraction of limpet and bacterial cores show no evidence of long-range crystallographic order. Chemical analysis indicates a high inorganic phosphate (Pi) (Fe/Pi = 1.71) content in bacterial ferritin compared with human ferritin (thalassemic) (Fe/Pi = 21.0). The high Pi content of bacterial ferritin suggests a hydrated amorphous iron (III) phosphate mineral core. Structural disorder within the limpet and bacterial cores may be associated with increased Pi content and increased oxidation in Fe(II), resulting in rapid mineral deposition. Growth of the iron (III) oxide cores in human ferritin is discussed on the basis of high resolution electron microscopy results.
A murine monoclonal antibody (OKT9) raised against human leukemic cells binds to a wide variety of leukemia and tumor cell lines and to a minority of leukemia cells taken directly from patients. Fetal thymus and liver are strongly reactive as are some normal, immature hemopoietic cells and activated lymphocytes. Reactivity with OKT9 appears to correlate with proliferation status in both normal and malignant populations. Biochemical analysis indicates that this structure is a approximately equal to 180,000-dalton glycoprotein with two disulfide-bonded subunits of approximately equal to 90,000-daltons. Isolation of the transferrin receptor from a T-cell line (MOLT-4) indicates that it also has a dimeric approximately equal to 180,000-dalton structure. Radio-labeled transferrin bound to its receptors can be specifically precipitated by the monoclonal OKT9, although the latter does not bind transferrin itself, indicating that the antigenic structure defined by this antibody is likely to be the transferrin receptor.
A monoclonal antibody highly specific for benign and malignant hepatocytes (HepPar 1) was evaluated as part of an antibody panel used to differentiate hepatocellular from nonhepatocellular neoplasms. Sixty-five liver tumors and two extrahepatic tumors from patients with documented liver tumors were studied. Twenty-two neoplasms were of hepatocellular origin, three were combined hepatocellular/cholangiocarcinomas, and the remainder were of nonhepatocellular origin. HepPar 1 alone had an 82% sensitivity and 90% specificity for the detection of hepatocellular neoplasms. The corresponding values for alpha-fetoprotein were 57% and 97%. Polyclonal antibody to carcinoembryonic antigen (canalicular pattern) had a sensitivity of 79% and specificity of 97% for these tumors. The use of antibody panels provided superior results when compared with individual antibodies. In summary, HepPar 1 monoclonal antibody is a useful reagent for the differential diagnosis of hepatocellular tumors. Its utility is enhanced when it is used as part of a diagnostic antibody panel.
We describe a strategy for the creation of recombinant ferritin heteropolymers which mimic the natural heterogeneity of this protein. This method entailed the co-expression of cDNA for both ferritin H and ferritin L subunits in a single bacterium using either a bicistronic vector, in which both cDNAs were expressed from the vector, or a dual vector expression strategy, in which each subunit was expressed from a separate compatible plasmid in a single bacterial host. Electron microscopy and sucrose density gradient centrifugation demonstrated that ferritin assembled spontaneously in such bacteria to form catalytically active proteins of the expected size and shape. Isoelectric focusing revealed that protein isolated from any of these bacteria exhibited a restricted heterogeneity in subunit composition. Such multi-subunit recombinant ferritins spontaneously assembled in bacteria may be useful in further studies of ferritin assembly and function. Our results further suggest that varying expression levels is a simple way to alter levels of individual components within a multi-subunit recombinant protein, and that this approach may be of general utility in assessing the contribution of individual components to the function of multi-subunit proteins or protein complexes.
Distinguishing hepatocellular carcinoma (HCC) from metastatic adenocarcinoma (MA) and cholangiocarcinoma (CC) can, at times, be difficult and sometimes requires immunohistochemical analysis. Recently, MOC31, an antibody directed against a cell surface glycoprotein, has been shown to be useful in separating HCC from both MA and CC; however, no study has compared MOC31 and other frequently used immunostains. We compare MOC31 with other commonly used immunostains for HCC, MA, and CC. Formalin-fixed, paraffin-embedded tissue sections from 57 previously characterized hepatic neoplasms (13 HCC, 14 CC, 3 combined HCC-CC, and 27 MA) were immunostained with antibodies directed against MOC31, cytokeratin (CK) 7, CK20, alpha-fetoprotein (AFP), polyclonal carcinoembryonic antigen, Ber-EP4, and Factor XIII-A. Two pathologists reviewed slides, and positivity was defined as more than 1% of cells staining with the appropriate pattern. Positive MOC31 immunostaining was seen in 0 of 13 HCC, 13 of 14 CC, 3 of 3 HCC-CC, and 27 of 27 MA; the staining was strong and diffuse. CK20 reactivity was observed in 0 of 13 HCC, 2 of 14 CC, 0 of 3 HCC-CC, and 12 of 27 MA; CK7 immunostained 4 of 13 HCC, 13 of 14 CC, 3 of 3 HCC-CC, and 15 of 27 MA; AFP was detected in 4 of 13 HCC and 2 of 3 HCC-CC, whereas all CC and MA were negative; polyclonal carcinoembryonic antigen showed immunoreactivity in 12 of 13 HCC and 3 of 3 HCC-CC in a canalicular pattern, whereas diffuse positivity was identified in 13 of 14 CC and 26 of 27 MA; Ber-EP4 immunostained 1 of 13 HCC, 14 of 14 CC, 2 of 3 HCC-CC, and 26 of 27 MA; and Factor XIII-A was negative in all HCC, CC, and MA. MOC31 expression distinguished HCC from adenocarcinoma in 56 of 57 cases. AFP was specific for HCC but was not sensitive. CK7 and CK20 have limited utility in distinguishing HCC from CC or MA, and Factor XIII-A is not useful. Ber-EP4 staining was similar to MOC31, but one HCC did stain with Ber-EP4. Polyclonal CEA yields similar numerical results as MOC31, but the focal nature of the staining and occasional difficulty in evaluating the pattern can make interpretation problematic. We conclude that MOC31 should be a component of the immunohistochemical panel to distinguish HCC from CC and MA.
Hepatocyte monoclonal antibody (Hep) (alternatively Hep Par 1 for Hep paraffin 1) has been reported to stain normal hepatic tissue and hepatocellular carcinoma (HCC) with high specificity. We have studied the Hepatocyte expression in 96 cases of HCC and 311 cases of nonhepatic epithelial tumors. All cases of HCC were also stained with CEA-Gold 5, CD10, and alpha-fetoprotein. Hep, CEA-Gold 5, CD10, and alpha-fetoprotein immunostains were performed on formalin-fixed, paraffin-embedded tissue sections. Hep immunoreactivity was detected in 88 of 96 cases of HCC (92%), with a cytoplasmic and granular pattern of staining. The level of Hep expression in HCC corresponded to the nuclear grade and growth pattern. All 50 cases of nuclear grade 1 and nuclear grade 2 HCC were positive (100%), whereas 37 of 44 nuclear grade 3 (84%) and 1 of 2 nuclear grade 4 (50%) were positive. Sixty-seven of 68 cases of HCC with a trabecular, pseudoglandular, or scirrhous growth pattern were positive (98%), whereas 22 of 27 cases of HCC with a compact growth pattern were positive (81%). CEA-Gold 5, CD10, and alpha-fetoprotein immunoreactivity was detected in 76% (73 of 96), 52% (50 of 96), and 31% (30 of 96) cases of HCC, respectively. The positive predictive value of the combination of all four antibodies was 97%. Three cases of HCC were negative for all four antibodies; these cases had a high nuclear grade or a sarcomatoid or compact growth pattern. Twenty of 311 cases of nonhepatic tumors were positive for Hep (6%): 15 were adenocarcinomas and five were neuroendocrine tumors. The negative predictive value of Hep in HCC was 94%. The Hep-positive nonhepatic epithelial tumors were easily distinguished from HCC by the expression of keratin 7 or keratin 20 for adenocarcinoma and chromogranin and synaptophysin for neuroendocrine tumors because HCC does usually not express these markers. With the exception of two cases of hepatoid gastric carcinoma, all Hep-positive nonhepatic epithelial tumors were negative for alpha-fetoprotein, CEA-Gold 5, and CD10. Our study demonstrates that Hep is a relatively specific marker for HCC. It is useful in differentiating HCC from primary hepatic cholangiocarcinoma and metastatic tumors when combined with other immunomarkers.
Early noninvasive detection and characterization of solid tumors and their supporting neovasculature is a fundamental prerequisite for effective therapeutic intervention, particularly antiangiogenic treatment regimens. Emerging molecular imaging techniques now allow recognition of early biochemical, physiological, and anatomical changes before manifestation of gross pathological changes. Although new tumor, vascular, extracellular matrix, and lymphatic biomarkers continue to be discovered, the alpha(nu)beta(3)-integrin remains an attractive biochemical epitope that is highly expressed on activated neovascular endothelial cells and essentially absent on mature quiescent cells. In this study, we report the first in vivo use of a magnetic resonance (MR) molecular imaging nanoparticle to sensitively detect and spatially characterize neovascularity induced by implantation of the rabbit Vx-2 tumor using a common clinical field strength (1.5T). New Zealand White rabbits (2 kg) 12 days after implantation of fresh Vx-2 tumors (2 x 2 x 2 mm(3)) were randomized into one of three treatment groups: (a) alpha(nu)beta(3)-targeted, paramagnetic formulation; (b) nontargeted, paramagnetic formulation; and (c) alpha(nu)beta(3)-targeted nonparamagnetic nanoparticles followed by (2 h) the alpha(nu)beta(3)-targeted, paramagnetic formulation to competitively block magnetic resonance imaging (MRI) signal enhancement. After i.v. systemic injection (0.5 ml of nanoparticles/kg), dynamic T(1)-weighted MRI was used to spatially and temporally determine nanoparticle deposition in the tumor and adjacent tissues, including skeletal muscle. At 2-h postinjection, alpha(nu)beta(3)-targeted paramagnetic nanoparticles increased MRI signal by 126% in asymmetrically distributed regions primarily in the periphery of the tumor. Similar increases in MR contrast were also observed within the walls of some vessels proximate to the tumor. Despite their relatively large size, nanoparticles penetrated into the leaky tumor neovasculature but did not appreciably migrate into the interstitium, leading to a 56% increase in MR signal at 2 h. Pretargeting of the alpha(nu)beta(3)-integrin with nonparamagnetic nanoparticles competitively blocked the specific binding of alpha(nu)beta(3)-targeted paramagnetic nanoparticles, decreasing the MR signal enhancement (50%) to a level attributable to local extravasation. The MR signal of adjacent hindlimb muscle or contralateral control tissues was unchanged by either the alpha(nu)beta(3)-targeted or control paramagnetic agents. Immunohistochemistry of alpha(nu)beta(3)-integrin corroborated the extent and asymmetric distribution of neovascularity observed by MRI. These studies demonstrate the potential of this targeted molecular imaging agent to detect and characterize (both biochemically and morphologically) early angiogenesis induced by minute solid tumors with a clinical 1.5 Tesla MRI scanner, facilitating the localization of nascent cancers or metastases, as well as providing tools to phenotypically categorize and segment patient populations for therapy and to longitudinally follow the effectiveness of antitumor treatment regimens.
Transferrin receptor (TFRC) is a membrane-bound protein expressed in larger amounts in proliferating, e.g., malignant, cells than in quiescent cells. The specific expression of TFRC can represent a diagnostic tool or a therapeutic target in solid tumours expressing this antigen. Whether TFRC is expressed in human pancreatic tumours is unknown. The aim of this study was the investigation of the expression of TFRC and transferrin in human pancreatic cancer and in neuroendocrine tumours of the pancreas. Fifty one specimens of human pancreatic cancer and 14 samples of pancreatic neuroendocrine tumours were obtained after surgery. The expression of TFRC, transferrin and cytokeratin was studied by standard immunohistochemistry. Flow cytometry was used for the investigation of TFRC expression in nine cell lines of ductal pancreatic cancer in vitro. In contrast to normal tissue, 93% of pancreatic tumour cells showed positive (82%) or heterogeneous (11%) expression of TFRC. It was strongly expressed by malignant epithelial cells; normal stromal and endothelial cells were not stained by anti-TFRC antibodies. Primary tumours and metastases showed a similar frequency of TFRC expression. Three neuroendocrine carcinomas showed positive expression of TFRC by malignant tumour cells. The expression of TFRC was negative in benign neuroendocrine tumours of the pancreas. The cell lines of pancreatic cancer were characterised by a low expression of TFRC in vitro. In contrast to normal pancreatic tissue and benign neuroendocrine tumours of the pancreas, pancreatic cancer and neuroendocrine carcinoma are therefore characterised frequently by high expression of TFRC. Hence, TFRC represents a marker of malignant transformation in the pancreas that could be applied as potential diagnostic and therapeutic target.
Thyroid transcription factor-1 (TTF-1) is considered as a reliable marker for differential diagnosis in distinguishing primary adenocarcinomas of the lung from extrathoracic origins. We previously reported the first case of lung metastasis of colorectal origin, with nuclear expression of TTF-1. As most previous studies were performed on series of extrathoracic primary tumors, we raised the question of a possible role of lung microenviroment in TTF-1 expression. We investigated the rate of TTF-1 expression in lung metastases of extrathoracic adenocarcinomas and compared results of immunohistochemistry performed with different primary antibodies. Two different clones of antibodies (8G7G1/1 from Dako, SPT24 from Novocastra) raised against TTF-1 were used on 56 lung-metastatic malignant tumors, 41 from colorectal origin. A series of primary colorectal (90 cases) and primary pulmonary adenocarcinomas (86 cases) were also investigated. Four of 41 (10%) lung metastases of colorectal adenocarcinomas displayed a nuclear staining for TTF-1 with SPT24 clone. Three of the four positive cases displayed similar nuclear staining in primary and/or other extrathoracic metastatic sites as well as four of 90 (5%) primary colorectal adenocarcinomas, ruling out the role of lung microenvironment. None of them was positive with 8G7G1/1 clone. Sensitivity between two sets of antibodies was compared in 86 primary pulmonary adenocarcinomas. Nuclear staining was detected in 72 cases (84%) with Novocastra's antibody and 56 cases (65%) with Dako's. Significant discordance was observed (P < 0.01). These results suggest that the diagnostic virtue of TTF-1 detection depends on the used antibody's clone. The SPT24 clone seems to have a stronger affinity for TTF-1 protein but may lead to a few positive colorectal adenocarcinomas.
In this article, a systematic study of the design and development of surface-modification schemes for silica nanoparticles is presented. The nanoparticle surface design involves an optimum balance of the use of inert and active surface functional groups to achieve minimal nanoparticle aggregation and reduce nanoparticle nonspecific binding. Silica nanoparticles were prepared in a water-in-oil microemulsion and subsequently surface modified via cohydrolysis with tetraethyl orthosilicate (TEOS) and various organosilane reagents. Nanoparticles with different functional groups, including carboxylate, amine, amine/phosphonate, poly(ethylene glycol), octadecyl, and carboxylate/octadecyl groups, were produced. Aggregation studies using SEM, dynamic light scattering, and zeta potential analysis indicate that severe aggregation among amine-modified silica nanoparticles can be reduced by adding inert functional groups, such as methyl phosphonate, to the surface. To determine the effect of various surface-modification schemes on nanoparticle nonspecific binding, the interaction between functionalized silica nanoparticles and a DNA chip was also studied using confocal imaging/fluorescence microscopy. Dye-doped silica nanoparticles functionalized with octadecyl and carboxylate groups showed minimal nonspecific binding. Using these surface-modification schemes, fluorescent dye-doped silica nanoparticles can be more readily conjugated with biomolecules and used as highly fluorescent, sensitive, and reproducible labels in bioanalytical applications.
We have developed the chemistry for preparing a universal probe and the appropriate nano- and microparticle labels that can be used to do highly selective multiplexed detection of three protein cancer markers at low-femtomolar concentration in buffer and serum media. The approach relies on a new multiplexed version of the biobarcode amplification method and offers new opportunities for studying multiple protein markers in a single sample. This could lead to new forms of disease diagnosis and monitoring disease recurrence in a variety of settings.
The transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and in the regulation of cell growth. Iron uptake occurs via the internalization of iron-loaded transferrin (Tf) mediated by the interaction with the TfR. In addition, the TfR may also contain other growth regulatory properties in certain normal and malignant cells. The elevated levels of TfR in malignancies, its relevance in cancer, and the extracellular accessibility of this molecule make it an excellent antigen for the treatment of cancer using antibodies. The TfR can be targeted by monoclonal antibodies specific for the extracellular domain of the receptor. In this review, we summarize advancements in the basic physiology of the TfR including structure, function, and expression. We also discuss the efficacy of targeting the TfR using cytotoxic antibodies that inhibit cell growth and/or induce apoptosis in targeted malignant cells.
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.
We developed a new imaging probe for atherosclerotic lesion imaging by chemically conjugating an atherosclerotic plaque-homing peptide (termed the AP peptide) to hydrophobically modified glycol chitosan (HGC) nanoparticles. The AP peptide was previously discovered by using an in vivo phage display screening method. HGC nanoparticles were labeled with the near-infrared (NIR) fluorophore Cy5.5, yielding nanoparticles 314 nm in diameter. The binding characteristics of nanoparticles to cytokine (TNF-alpha)-activated bovine aortic endothelial cells (BAECs) were studied in vitro under static conditions and in a dynamic flow environment. AP-tagged HGC-Cy5.5 nanoparticles (100 microg/ml, 2 h incubation) bound more avidly to TNF-alpha-activated BAECs than to unactivated BAECs. Nanoparticles were mostly located in the membranes of BAECs, although some were taken up by the cells and were visible in the cytoplasm, suggesting that the AP peptides in HGC nanoparticles retained target selectivity for activated BAECs. Binding selectivity of AP-tagged HGC-Cy5.5 nanoparticles was also studied in vivo. NIR fluorescence imaging demonstrated that AP-tagged HGC-Cy5.5 nanoparticles bound better to atherosclerotic lesions in a low-density lipoprotein receptor-deficient (Ldlr(-/-)) atherosclerotic mouse than to such lesions in a normal mouse. These results suggest that the newly designed AP-tagged HGC-Cy5.5 nanoparticles may be useful for atherosclerotic lesion imaging, and may also be employed to elucidate pathophysiological changes, at the molecular level, on atherosclerotic endothelium.
The Diagnostic Value of Immunohistochemistry in the Diagnosis of Primary and Secondary Hepatic Carcinomas
- Ali S Sawan
Ali S. Sawan. The Diagnostic Value of Immunohistochemistry in the Diagnosis of Primary and Secondary Hepatic Carcinomas. JKAU: Med. Sci. 16, 37-48(2009).
Cell-specific targeting of nanoparticles by multivalent attachment of small molecules
- R Weissleder
- K Kelly
- E Y Sun
- T Shtatland
- L Josephson
Weissleder, R., Kelly, K., Sun, E. Y., Shtatland, T. & Josephson, L. Cell-specific
targeting of nanoparticles by multivalent attachment of small molecules. Nature
Biotechnol. 23, 1418-1423 (2005).
Molecular imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel ανβ3-targeted nanoparticle and 1.5 tesla magnetic resonance imaging
- P M Winter
- PM Winter