Effect of Gold Nanostars Plus Amikacin against Carbapenem-Resistant Klebsiella pneumoniae Biofilms.

(1) Background: Carbapenem-resistant Klesiella pneumoniae (CR-KP) infection rates depict an almost pre-antibiotic scenario since the pipeline for effective antibiotics against this pathogen has been almost entirely depleted. This study aims to evaluate the antibacterial effect of gold nanostars (GNS) alone or associated with some of the most widely used antibiotics for the treatment of CR-KP strains, i.e., meropenem or amikacin, on both planktonic and sessile forms. Additionally, we measured the effect of GNS on cell proliferation and biocompatibility in invertebrate in vivo models. (2) Materials and methods: GNS were made from gold seeds grown using a seeded-growth surfactant-free method assisted by silver ions and functionalized with mercapto-poly(ethylene glycol)amino by ligand exchange. The antimicrobial capacity, effect on cell proliferation, and biocompatibility of the most effective combination was evaluated in a Galleria mellonella model. (3) Results: The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 80 and 160 µM of GNS for all strains, respectively. The minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were >320 µM of GNS for both. A synergy was found between GNS and amikacin. Larvae administered GNS plus amikacin were found to tolerate the treatment well, which prevented infection. (4) Conclusions: GNS are a promising anti-CR-KP nanomaterial.

Bacteria and cells as alternative nano-carriers for biomedical applications.

INTRODUCTION: Nano-based systems have received a lot of attention owing to their particular properties and, hence, have been proposed for a wide variety of biomedical applications. These nanosystems could be potentially employed for diagnosis and therapy of different medical issues. Although these nanomaterials are designed for specific tasks, interactions, and transformations when administered to the human body affect their performance and behavior. In this regard, bacteria and other cells have been presented as alternative nanocarriers. These microorganisms can be genetically modified and customized for a more specific therapeutic action and, in combination with nanomaterials, can lead to bio-hybrids with a unique potential for biomedical purposes. AREAS COVERED: Literature regarding bacteria and cells employed in combination with nanomaterials for biomedical applications is revised and discussed in this review. The potential as well as the limitations of these novel bio-hybrid systems are evaluated. Several examples are presented to show the performance of these alternative nanocarriers. EXPERT OPINION: Bio-hybrid systems have shown their potential as alternative nanocarriers as they contribute to better performance than traditional nano-based systems. Nevertheless, their limitations must be studied, and advantages and drawbacks assessed before their application to medicine.

Hard and Soft Protein Corona of Nanomaterials: Analysis and Relevance.

Upon contact with a biological milieu, nanomaterials tend to interact with biomolecules present in the media, especially proteins, leading to the formation of the so-called "protein corona". As a result of these nanomaterial-protein interactions, the bio-identity of the nanomaterial is altered, which is translated into modifications of its behavior, fate, and pharmacological profile. For biomedical applications, it is fundamental to understand the biological behavior of nanomaterials prior to any clinical translation. For these reasons, during the last decade, numerous publications have been focused on the investigation of the protein corona of many different types of nanomaterials. Interestingly, it has been demonstrated that the structure of the protein corona can be divided into hard and soft corona, depending on the affinity of the proteins for the nanoparticle surface. In the present document, we explore the differences between these two protein coronas, review the analysis techniques used for their assessment, and reflect on their relevance for medical purposes.

Thermal monitoring during photothermia: hybrid probes for simultaneous plasmonic heating and near-infrared optical nanothermometry.

The control of temperature during photothermal therapy is key to preventing unwanted damage in surrounding tissue or post-treatment inflammatory responses. Lack of accurate thermal control is indeed one of the main limitations that hyperthermia techniques present to allow their translation into therapeutic applications. We developed a nanoprobe that allows controlled local heating, combined with in situ nanothermometry. The design of the probe follows a practical rationale that aims at simplifying experimental requirements and exploits exclusively optical wavelengths matching the first and second biological windows in the near-infrared. Methods: Hybrid nanostructures were chemically synthesized, and combine gold nanostars (photothermal agents) with CaF2:Nd3+,Y3+ nanoparticles (luminescent nanothermometers). Both components were simultaneously excited in the near-infrared range, at 808 nm. Following the goal of simplifying the thermal monitoring technique, the luminescent signal was recorded with a portable near-infrared detector. The performance of the probes was tested in 3D tumor spheroids from a human glioblastoma (U87MG) cell line. The location of the beads within the spheroids was determined measuring Nd3+ emission in a commercial Lightsheet microscope, modified in-house to be able to select the required near-infrared wavelengths. The temperature achieved inside the tumor spheroids was deduced from the luminescence of Nd3+, following a protocol that we developed to provide reliable thermal readings. Results: The choice of materials was shown to work as an optically excited hybrid probe. Depending on the illumination parameters, temperature can be controlled in a range between 37 ºC and 100 ºC. The near-infrared emission of nanothermometers also allows microscopic tracking of the hybrid nanostructures, confirming that the probes can penetrate deeper into the spheroid mass. We observed that, application of optical thermometry in biological environments requires often neglected considerations, since the optical signal changes along the optical path. Accordingly, we developed data analysis protocols that guarantee reliable thermal readings. Conclusions: The prepared hybrid probes are internalized in 3D tumor spheroids and can be used to induce cell death through photothermal effects, while simultaneously measuring the local temperature in situ. We show that luminescent thermometry in biomedical applications requires the development of protocols that guarantee accurate readings. Regarding photothermal treatments, we observe a sharp thermal threshold at around 55 ºC (for 10 min treatments) that separates high survival ratio from complete cell death.

In vivo formation of protein corona on gold nanoparticles. The effect of their size and shape.

The efficacy of drug delivery and other nanomedicine-related therapies largely relies on the ability of nanoparticles to reach the target organ. However, when nanoparticles are injected into the bloodstream, their surface is instantly modified upon interaction with blood components, principally with proteins. It is well known that a dynamic and multi-layered protein structure is formed spontaneously on the nanoparticle upon contact with physiological media, which has been termed protein corona. Although several determinant factors involved in protein corona formation have been identified from in vitro studies, specific relationships between the nanomaterial synthetic identity and its ensuing biological identity under realistic in vivo conditions remain elusive. We present here a detailed study of in vivo protein corona formation after blood circulation of anisotropic gold nanoparticles (nanorods and nanostars). Plasmonic gold nanoparticles of different shapes and sizes were coated with polyethyleneglycol, intravenously administered in CD-1 mice and subsequently recovered. The results from gel electrophoresis and mass spectrometry analysis revealed the formation of complex protein coronas, as early as 10 minutes post-injection. The total amount of protein adsorbed onto the particle surface and the protein corona composition were found to be affected by both the particle size and shape.

3D scaffold with effective multidrug sequential release against bacteria biofilm.

Bone infection is a feared complication following surgery or trauma that remains as an extremely difficult disease to deal with. So far, the outcome of therapy could be improved with the design of 3D implants, which combine the merits of osseous regeneration and local multidrug therapy so as to avoid bacterial growth, drug resistance and the feared side effects. Herein, hierarchical 3D multidrug scaffolds based on nanocomposite bioceramic and polyvinyl alcohol (PVA) prepared by rapid prototyping with an external coating of gelatin-glutaraldehyde (Gel-Glu) have been fabricated. These 3D scaffolds contain three antimicrobial agents (rifampin, levofloxacin and vancomycin), which have been localized in different compartments of the scaffold to obtain different release kinetics and more effective combined therapy. Levofloxacin was loaded into the mesopores of nanocomposite bioceramic part, vancomycin was localized into PVA biopolymer part and rifampin was loaded in the external coating of Gel-Glu. The obtained results show an early and fast release of rifampin followed by sustained and prolonged release of vancomycin and levofloxacin, respectively, which are mainly governed by the progressive in vitro degradability rate of these scaffolds. This combined therapy is able to destroy Gram-positive and Gram-negative bacteria biofilms as well as inhibit the bacteria growth. In addition, these multifunctional scaffolds exhibit excellent bioactivity as well as good biocompatibility with complete cell colonization of preosteoblast in the entire surface, ensuring good bone regeneration. These findings suggest that these hierarchical 3D multidrug scaffolds are promising candidates as platforms for local bone infection therapy. STATEMENT OF SIGNIFICANCE: The present study is focused in finding an adequate therapeutic solution for the treatment of bone infection based on 3D multifunctional scaffolds, which combines the merits of osseous regeneration and local multidrug delivery. These 3D multidrug scaffolds, containing rifampin, levofloxacin and vancomycin, localized in different compartments to achieve different release kinetics. These 3D multidrug scaffolds displays an early and fast release of rifampin followed by sustained and prolonged release of vancomycin and levofloxacin, which are able to destroy Staphylococcus and Escherichia biofilms as well as inhibit bacteria growth in very short time periods. This new combined therapy approach involving the sequential delivery of antibiofilms with antibiotics constitutes an excellent and promising alternative for bone infection treatment.