When combined with immunotherapy, image-guided targeted delivery of chemotherapeutic agents is a promising direction for combination cancer theranostics, but this approach has so far produced only ...limited success due to a lack of molecular targets on the cell surface and low therapeutic index of conventional chemotherapy drugs. Here, we demonstrate a synergistic strategy of combination immuno/chemotherapy in conditions of dual regioselective targeting, implying vectoring of two distinct binding sites of a single oncomarker (here, HER2) with theranostic compounds having a different mechanism of action. We use: (i) PLGA nanoformulation, loaded with an imaging diagnostic fluorescent dye (Nile Red) and a chemotherapeutic drug (doxorubicin), and functionalized with affibody ZHER2:342 (8 kDa); (ii) bifunctional genetically engineered DARP-LoPE (42 kDa) immunotoxin comprising of a low-immunogenic modification of therapeutic Pseudomonas exotoxin A (LoPE) and a scaffold targeting protein, DARPin9.29 (14 kDa). According to the proposed strategy, the first chemotherapeutic nanoagent is targeted by the affibody to subdomain III and IV of HER2 with 60-fold specificity compared with nontargeted particles, while the second immunotoxin is effectively targeted by DARPin molecule to subdomain I of HER2. We demonstrate that this dual targeting strategy can enhance anticancer therapy of HER2-positive cells with a very strong synergy, which made possible 1000-fold decrease of effective drug concentration in vitro and a significant enhancement of HER2 cancer therapy compared to monotherapy in vivo. Moreover, this therapeutic combination prevented the appearance of secondary tumor nodes. Thus, the suggested synergistic strategy utilizing dual targeting of the same oncomarker could give rise to efficient methods for aggressive tumors treatment.
Nanostructured materials and systems find various applications in biomedical fields. Hybrid organo-inorganic nanomaterials are intensively studied in a wide range of areas, from visualization to drug ...delivery or tissue engineering. One of the recent trends in material science is biomimetic approaches toward the synthesis or modification of functional nanosystems. Here, we describe an approach toward multifunctional nanomaterials through the biomimetic polymerization of dopamine derivatives. Magnetite nanoparticles were modified with a combination of dopamine conjugates to give multifunctional magneto-fluorescent nanocomposites in one synthetic step. The obtained material showed excellent biocompatibility at concentrations up to 200 μg/mL and an in vivo biodistribution profile typical for nanosized formulations. The synthesized systems were conjugated with antibodies against HER2 to improve their selectivity toward HER2-positive cancer cells. The produced material can be used for dual magneto-optical in vivo studies or targeted drug delivery. The applied synthetic strategy can be used for the creation of various multifunctional hybrid nanomaterials in mild conditions.
Nanoparticles exhibiting the localized surface plasmon resonance (LSPR) phenomenon are promising tools for diagnostics and cancer treatment. Among widely used metal nanoparticles, silver ...nanoparticles (Ag NPs) possess the strongest light scattering and surface plasmon strength. However, the therapeutic potential of Ag NPs has until now been underestimated. Here we show targeted photothermal therapy of solid tumors with 35 nm HER2-targeted Ag NPs, which were produced by the green synthesis using an aqueous extract of
Mill. Light irradiation tests demonstrated effective hyperthermic properties of these NPs, namely heating by 10 °C in 10 min. To mediate targeted cancer therapy, Ag NPs were conjugated to the scaffold polypeptide, affibody Z
, which recognizes a clinically relevant oncomarker HER2. The conjugation was mediated by the PEG linker to obtain Ag-PEG-HER2 nanoparticles. Flow cytometry tests showed that Ag-PEG-HER2 particles successfully bind to HER2-overexpressing cells with a specificity comparable to that of full-size anti-HER2 IgGs. A confocal microscopy study showed efficient internalization of Ag-PEG-HER2 into cells in less than 2 h of incubation. Cytotoxicity assays demonstrated effective cell death upon exposure to Ag-PEG-HER2 and irradiation, caused by the production of reactive oxygen species. Xenograft tumor therapy with Ag-PEG-HER2 particles in vivo resulted in full primary tumor regression and the prevention of metastatic spread. Thus, for the first time, we have shown that HER2-directed plasmonic Ag nanoparticles are effective sensitizers for targeted photothermal oncotherapy.
Therapy for aggressive metastatic breast cancer remains a great challenge for modern biomedicine. Biocompatible polymer nanoparticles have been successfully used in clinic and are seen as a potential ...solution. Specifically, researchers are exploring the development of chemotherapeutic nanoagents targeting the membrane-associated receptors of cancer cells, such as HER2. However, there are no targeting nanomedications that have been approved for human cancer therapy. Novel strategies are being developed to alter the architecture of agents and optimize their systemic administration. Here, we describe a combination of these approaches, namely, the design of a targeted polymer nanocarrier and a method for its systemic delivery to the tumor site. Namely, PLGA nanocapsules loaded with a diagnostic dye, Nile Blue, and a chemotherapeutic compound, doxorubicin, are used for two-step targeted delivery using the concept of tumor pre-targeting through the barnase/barstar protein "bacterial superglue". The first pre-targeting component consists of an anti-HER2 scaffold protein, DARPin9_29 fused with barstar, Bs-DARPin9_29, and the second component comprises chemotherapeutic PLGA nanocapsules conjugated to barnase, PLGA-Bn. The efficacy of this system was evaluated in vivo. To this aim, we developed an immunocompetent BALB/c mouse tumor model with a stable expression of human HER2 oncomarkers to test the potential of two-step delivery of oncotheranostic nano-PLGA. In vitro and ex vivo studies confirmed HER2 receptor stable expression in the tumor, making it a feasible tool for HER2-targeted drug evaluation. We demonstrated that two-step delivery was more effective than one-step delivery for both imaging and tumor therapy: two-step delivery had higher imaging capabilities than one-step and a tumor growth inhibition of 94.9% in comparison to 68.4% for the one-step strategy. The barnase*barstar protein pair has been proven to possess excellent biocompatibility, as evidenced by the successful completion of biosafety tests assessing immunogenicity and hemotoxicity. This renders the protein pair a highly versatile tool for pre-targeting tumors with various molecular profiles, thereby enabling the development of personalized medicine.
The development of non-invasive photothermal therapy (PTT) methods utilizing nanoparticles as sensitizers is one of the most promising directions in modern oncology. Nanoparticles loaded with ...photothermal dyes are capable of delivering a sufficient amount of a therapeutic substance and releasing it with the desired kinetics in vivo. However, the effectiveness of oncotherapy methods, including PTT, is often limited due to poor penetration of sensitizers into the tumor, especially into solid tumors of epithelial origin characterized by tight cellular junctions. In this work, we synthesized 200 nm nanoparticles from the biocompatible copolymer of lactic and glycolic acid, PLGA, loaded with magnesium phthalocyanine, PLGA/Pht-Mg. The PLGA/Pht-Mg particles under the irradiation with NIR light (808 nm), heat the surrounding solution by 40 °C. The effectiveness of using such particles for cancer cells elimination was demonstrated in 2D culture in vitro and in our original 3D model with multicellular spheroids possessing tight cell contacts. It was shown that the mean inhibitory concentration of such nanoparticles upon light irradiation for 15 min worsens by more than an order of magnitude: IC50 increases from 3 µg/mL for 2D culture vs. 117 µg/mL for 3D culture. However, when using the JO-4 intercellular junction opener protein, which causes a short epithelial-mesenchymal transition and transiently opens intercellular junctions in epithelial cells, the efficiency of nanoparticles in 3D culture was comparable or even outperforming that for 2D (IC50 = 1.9 µg/mL with JO-4). Synergy in the co-administration of PTT nanosensitizers and JO-4 protein was found to retain in vivo using orthotopic tumors of BALB/c mice: we demonstrated that the efficiency in the delivery of such nanoparticles to the tumor is 2.5 times increased when PLGA/Pht-Mg nanoparticles are administered together with JO-4. Thus the targeting the tumor cell junctions can significantly increase the performance of PTT nanosensitizers.
Nanoparticle-based chemotherapy is considered to be an effective approach to cancer diagnostics and therapy in modern biomedicine. However, efficient tumor targeting remains a great challenge due to ...the lack of specificity, selectivity, and high dosage of chemotherapeutic drugs required. A two-step targeted drug delivery strategy (DDS), involving cancer cell pre-targeting, first with a first nontoxic module and subsequent targeting with a second complementary toxic module, is a solution for decreasing doses for administration and lowering systemic toxicity. To prove two-step DDS efficiency, we performed a direct comparison of one-step and two-step DDS based on chemotherapy loaded PLGA nanoparticles and barnase*barstar interface. Namely, we developed and thoroughly characterized the two-step targeting strategy of HER2-overexpressing cancer cells. The first targeting block consists of anti-HER2 scaffold polypeptide DARPin9_29 fused with barstar. Barstar exhibits an extremely effective binding to ribonuclease barnase with K
= 10
M
, thus making the barnase*barstar protein pair one of the strongest known protein*protein complexes. A therapeutic PLGA-based nanocarrier coupled to barnase was used as a second targeting block. The PLGA nanoparticles were loaded with diagnostic dye, Nile Blue, and a chemotherapeutic drug, doxorubicin. We showed that the two-step DDS increases the performance of chemotherapy-loaded nanocarriers: IC50 of doxorubicin delivered via two-step DDS was more than 100 times lower than that for one-step DDS: IC50 = 43 ± 3 nM for two-step DDS vs. IC50 = 4972 ± 1965 nM for one-step DDS. The obtained results demonstrate the significant efficiency of two-step DDS over the classical one-step one. We believe that the obtained data will significantly change the direction of research in developing targeted anti-cancer drugs and promote the creation of new generation cancer treatment strategies.
Photothermal therapy is one of the most promising and rapidly developing fields in modern oncology due to its high efficiency, localized action, and minimal invasiveness. Polymeric nanoparticles ...(NPs) incorporating low molecular-weight photothermal dyes are capable of delivering therapeutic agents to the tumor site, releasing them in a controlled manner, and providing tumor treatment under external light irradiation. The nanoparticle synthesis components are critically important factors that influence the therapeutically significant characteristics of polymeric NPs. Here, we show the impact of stabilizers and solvents used for synthesis on the properties of PLGA NPs for photothermal therapy. We synthesized PLGA nanocarriers using the microemulsion method and varied the nature of the solvent and the concentration of the stabilizer—namely, chitosan oligosaccharide lactate. A phthalocyanine-based photosensitizer, which absorbs light in the NIR window, was encapsulated in the PLGA NPs. When mQ water was used as a solvent and chitosan oligosaccharide lactate was used at a concentration of 1 g/L, the PLGA NPs exhibited highly promising photothermal properties. The final composite of the nanocarriers demonstrated photoinduced cytotoxicity against EMT6/P cells under NIR laser irradiation in vitro and was suitable for bioimaging.
Targeted nanoparticles of different origins are considered as new-generation diagnostic and therapeutic tools. However, there are no targeted drug formulations within the composition of nanoparticles ...approved by the FDA for use in the clinic, which is associated with the insufficient effectiveness of the developed candidates, the difficulties of their biotechnological production, and inadequate batch-to-batch reproducibility. Targeted protein self-assembling nanoparticles circumvent this problem since proteins are encoded in DNA and the final protein product is produced in only one possible way. We believe that the combination of the endless biomedical potential of protein carriers as nanoparticles and the standardized protein purification protocols will make significant progress in "magic bullet" creation possible, bringing modern biomedicine to a new level. In this review, we are focused on the currently existing platforms for targeted self-assembling protein nanoparticles based on transferrin, lactoferrin, casein, lumazine synthase, albumin, ferritin, and encapsulin proteins, as well as on proteins from magnetosomes and virus-like particles. The applications of these self-assembling proteins for targeted delivery in vitro and in vivo are thoroughly discussed, including bioimaging applications and different therapeutic approaches, such as chemotherapy, gene delivery, and photodynamic and photothermal therapy. A critical assessment of these protein platforms' efficacy in biomedicine is provided and possible problems associated with their further development are described.
The extreme aggressiveness and lethality of many cancer types appeal to the problem of the development of new-generation treatment strategies based on smart materials with a mechanism of action that ...differs from standard treatment approaches. The targeted delivery of nanoparticles to specific cancer cell receptors is believed to be such a strategy; however, there are no targeted nano-drugs that have successfully completed clinical trials to date. To meet the challenge, we designed an alternative way to eliminate tumors in vivo. Here, we show for the first time that the targeting of lectin-equipped polymer nanoparticles to the glycosylation profile of cancer cells, followed by photodynamic therapy (PDT), is a promising strategy for the treatment of aggressive tumors. We synthesized polymer nanoparticles loaded with magnetite and a PDT agent, IR775 dye (mPLGA/IR775). The magnetite incorporation into the PLGA particle structure allows for the quantitative tracking of their accumulation in different organs and the performing of magnetic-assisted delivery, while IR775 makes fluorescent in vivo bioimaging as well as light-induced PDT possible, thus realizing the theranostics concept. To equip PLGA nanoparticles with targeting modality, the particles were conjugated with lectins of different origins, and the flow cytometry screening revealed that the most effective candidate for breast cancer cell labeling is ConA, a lectin from
. In vivo experiments showed that after i.v. administration, mPLGA/IR775-ConA nanoparticles efficiently accumulated in the allograft tumors under the external magnetic field; produced a bright fluorescent signal for in vivo bioimaging; and led to 100% tumor growth inhibition after the single session of PDT, even for large solid tumors of more than 200 mm
in BALB/c mice. The obtained results indicate that the mPLGA/IR775 nanostructure has great potential to become a highly effective oncotheranostic agent.
Cancer is unquestionably a global healthcare challenge, spurring the exporation of novel treatment approaches. In recent years, nanomaterials have garnered significant interest with the greatest ...hopes for targeted nanoformulations due to their cell-specific delivery, improved therapeutic efficacy, and reduced systemic toxicity for the organism. The problem of successful clinical translation of nanoparticles may be related to the fact that most in vitro tests are performed at pH values of normal cells and tissues, ranging from 7.2 to 7.4. The extracellular pH values of tumors are characterized by a shift to a more acidic region in the range of 5.6–7.0 and represent a crucial target for enhancing nanoparticle delivery to cancer cells. Here we show the method of non-active protein incorporation into the surface of HER2-targeted nanoparticles to achieve optimal cellular uptake within the pH range of the tumor microenvironment. The method efficacy was confirmed in vitro and in vivo showing the maximum binding of nanoparticles to cells at a pH value 6.4. Namely, fluorescent magnetic nanoparticles, modified with HER2-recognising affibody ZHER2:342, with proven specificity in terms of HER2 recognition (with 62-fold higher cellular uptake compared to control nanoparticles) were designed for targeting cancer cells at slightly acidic pH values. The stabilizing protein, namely, bovine serum albumin, one of the major blood components with widespread availability and biocompatibility, was used for the decoration of the nanoparticle surface to alter the pH response of the targeting magnetic conjugates. The optimally designed nanoparticles showed a bell-shaped dependency of interaction with cancer cells in the pH range of 5.6–8.0 with maximum cellular uptake at pH value 6.4 close to that of the tumor microenvironment. In vivo experiments revealed that after i.v. administration, BSA-decorated nanoparticles exhibited 2 times higher accumulation in tumors compared to magnetic nanoparticles modified with affibody only. Thus, we demonstrated a valid method for enhancing the specificity of targeted nanoparticle delivery to cancer cells without changing the functional components of nanoparticles.
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