Bacteria-targeting nanoparticles for managing infections

Thesis (Ph. D. in Chemical and Biomedical Engineering)--Harvard-MIT Program in Health Sciences and Technology, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.Bacterial infections continue to be a significant concern particularly in healthcare settings and in the developing world. Current challenges include the increasing spread of drug resistant (DR) organisms, the side effects of antibiotic therapy, the negative consequences of clearing the commensal bacterial flora, and difficulties in developing prophylactic vaccines. This thesis was an investigation of the potential of a class of polymeric nanoparticles (NP) to contribute to the management of bacterial infections. More specifically, steps were taken towards using these NPs (1) to achieve greater spatiotemporal control over drug therapy by more targeted antibiotic delivery to bacteria, and (2) to develop a prophylactic vaccine formulation against the common bacterial sexually transmitted disease (STD) caused by Chlamydia trachomatis. In the first part, we synthesized polymeric NPs containing poly(lactic-co-glycolic acid)- block-poly(L-histidine)-block-poly(ethylene glycol) (PLGA-PLH-PEG). We show that these NPs are able to bind to bacteria under model acidic infection conditions and are able to encapsulate and deliver vancomycin to inhibit the growth of Staphylococcus aureus bacteria in vitro. Further work showed that the PLGA-PLH-PEG-based NPs demonstrated the potential for competition for binding bacteria at a site of infection from soluble protein and model phagocytic and tissue-resident cells in a NP composition dependent manner. The NPs demonstrated low toxicity in vitro, were well tolerated by mice in vivo, and circulated in the blood on timescales comparable to control PLGA-PEG NPs. In the second part, we used PLGA-PLH-PEG-based NPs to design a prophylactic vaccine against the obligate intracellular bacterium Chlamydia trachomatis, the most common cause of bacterial STD in the world. Currently, no vaccines against this pathogen are approved for use in humans. We first formulated NPs encapsulating the TLR7 agonist R848 conjugated to poly(lactic acid) (R848-PLA) in PLGA-PLH-PEG-based NPs, then incubated these R848-NPs with UV-inactivated C. trachomatis bacteria in acidity, forming a construct. Mice immunized with this vaccine via genital or intranasal routes demonstrated protection from genital infection post immunization in a primarily CD4⁺ T cell-dependent manner. These results may suggest avenues for future work in designing and developing more targeted drug therapies or vaccine formulations for managing bacterial infections using polymeric nanoparticles.by Aleksandar Filip Radovic-Moreno.Ph.D.in Chemical and Biomedical Engineerin

POLLEN VIABILITY IN QUINCE CULTIVARS

Pollen viability of eight quince cultivars (ʻLeskovackaʼ, ʻVranjskaʼ, ʻMoravaʼ, ʻPazardzijskaʼ, ʻHemusʼ, ʻAsenicaʼ, ʻPortugalʼ and ʻTriumphʼ), was studied in the two-year period (2011-2012). Testing of pollen viability was performed using two methods: the staining of pollen with acetocarmine (indirect method) and pollen germination in vitro with sucrose and agar-agar (direct method). Studied cultivars differed significantly in terms of pollen viability. The lowest percentage of stained pollen grains was detected in ʻLeskovackaʼ cultivar (70.29%) and the highest in the cultivars ʻAsenicaʼ, ʻHemusʼ and ʻTriumphʼ (over 90%). Similarly to that, the lowest percentage of pollen germination was obtained in ʻLeskovackaʼ cultivar (62.86%) and the highest in the cultivars ʻMoravaʼ, ʻAsenicaʼ, and ʻTriumphʼ (over 80%). With the exception of ʻPortugalʼ cultivar, the values of pollen viability determined by staining with acetocarmine were higher for 3-15% compared to the pollen germination in vitro. However, values obtained using these tho methods are highly positively correlated. On the basis of obtained results, the both methods can be recommended as reliable tests for pollen viability of quince, although priority should be given to the method of pollen germination in vitro, because it is more accurate. All tested cultivars are distinguished for high pollen viability, and can be successfully used as male parents in hybridization. In addition, they also can be recommended as a good pollenisers when are planting new quince orchards

Influence of temperature and exploitation period on fatigue crack growth parameters in different regions of welded joints

-The influence of exploitation period and temperature on the fatigue crack growth parameters indifferent regions of a welded joint is analysed for new and exploited low-alloyed Cr-Mo steel A-387 Gr. B. Theparent metal is a part of a reactor mantle which was exploited for over 40 years, and recently replaced with newmaterial. Fatigue crack growth parameters, threshold value Kth, coefficient C and exponent m, have beendetermined, both at room and exploitation temperature. Based on testing results, fatigue crack growth resistancein different regions of welded joint is analysed in order to justify the selected welding procedure specificatio

Surface Charge-Switching Polymeric Nanoparticles for Bacterial Cell Wall-Targeted Delivery of Antibiotics

Bacteria have shown a remarkable ability to overcome drug therapy if there is a failure to achieve sustained bactericidal concentration or if there is a reduction in activity in situ. The latter can be caused by localized acidity, a phenomenon that can occur as a result of the combined actions of bacterial metabolism and the host immune response. Nanoparticles (NP) have shown promise in treating bacterial infections, but a significant challenge has been to develop antibacterial NPs that may be suitable for systemic administration. Herein we develop drug-encapsulated, pH-responsive, surface charge-switching poly(d,l-lactic-co-glycolic acid)-b-poly(l-histidine)-b-poly(ethylene glycol) (PLGA-PLH-PEG) nanoparticles for treating bacterial infections. These NP drug carriers are designed to shield nontarget interactions at pH 7.4 but bind avidly to bacteria in acidity, delivering drugs and mitigating in part the loss of drug activity with declining pH. The mechanism involves pH-sensitive NP surface charge switching, which is achieved by selective protonation of the imidazole groups of PLH at low pH. NP binding studies demonstrate pH-sensitive NP binding to bacteria with a 3.5 ± 0.2- to 5.8 ± 0.1-fold increase in binding to bacteria at pH 6.0 compared to 7.4. Further, PLGA-PLH-PEG-encapsulated vancomycin demonstrates reduced loss of efficacy at low pH, with an increase in minimum inhibitory concentration of 1.3-fold as compared to 2.0-fold and 2.3-fold for free and PLGA-PEG-encapsulated vancomycin, respectively. The PLGA-PLH-PEG NPs described herein are a first step toward developing systemically administered drug carriers that can target and potentially treat Gram-positive, Gram-negative, or polymicrobial infections associated with acidity.National Institutes of Health (U.S.) (Grant CA151884)National Institutes of Health (U.S.) (Grant EB003647)Prostate Cancer Foundation (Award in Nanotherapeutics)United States. Dept. of Defense (Prostate Cancer Research Program PC 051156)MIT-Portugal ProgramNational Science Foundation (U.S.). Graduate Research FellowshipNational Institutes of Health (U.S.) (Office of the Director Grant DP2OD008435

Investigation of p-SCN-Bn-DOTA-trastuzumab labeled with radioactive and non-radioactive lutetium and yttrium: a crucial step for future applications

The significance lies in preparing stable, trastuzumab-immunoconjugate through the utilization of non-radioactive LuCl3 and YCl3, via p-SCN-Bn-DOTA. This approach is crucial to determine potential physicochemical alterations in immunoconjugate structure following metal binding. Post-conjugation, employing a 1:20 molar ratio, freeze-drying was performed to obtain stable immunoconjugates for subsequent analysis. Several chemical methods were employed to characterize antibody stability and retained immunoreactivity within the formulated immunoconjugates. Proof of protein integrity came from SDS-PAGE electrophoresis, with uniform fragment intensities (25 kDa for light chain, 50 kDa for heavy chain) indicating antibody non-degradation (1). IR and Raman spectroscopy verified secondary structural changes, with the presence of characteristic amide bands in both spectra indicating the retention of native secondary structure (2). Employing MALDI-TOF-MS, 4.9 p-SCN-Bn-DOTA molecules were determined per antibody molecule. The promising outcomes from non-radioactive labeling provide an opportunity for potential labeling with radioactive lutetium-177 and yttrium-90, each with a specific activity of 200 µCi/mL. Radioisotopes were incubated with p-SCN-Bn-DOTA-trastuzumab for an hour at 40 ºC. Evaluation of radiochemical purity and stability was conducted using the ITLC-SG system. Optimal mobile phases, specifically 0.4 M methanol:sodium acetate (1:1) for yttrium-90 and 0.9% NaCl for lutetium-177, facilitated thorough examination. Remarkable radiolabeling efficiency was achieved, >96% for yttrium-90 and >99% for lutetium-177. Stability assessments after 72 hours demonstrated greater stability in 177Lu-p-SCN-Bn-DOTA-trastuzumab (<1.5% lutetium-177 release) compared to the 90Y-labeled counterpart (<17% yttrium-90 release). This study demonstrates the successful development of radioimmunoconjugates, positioning this agent for potential application in vivo investigations

HER-2-Targeted Nanoparticle-Affibody Bioconjugates for Cancer Therapy

Affibodies are a class of polypeptide ligands that are potential candidates for cell- or tissue-specific targeting of drug-encapsulated controlled release polymeric nanoparticles (NPs). Here we report the development of drug delivery vehicles comprised of polymeric NPs that are surface modified with Affibody ligands that bind to the extracellular domain of the trans-membrane human epidermal growth factor receptor 2 (HER-2) for targeted delivery to cells which over express the HER-2 antigen. NPs lacking the anti-HER-2 Affibody did not show significant uptake by these cells. Using paclitaxel encapsulated NP-Affibody (1 wt% drug loading), we demonstrated increased cytotoxicity of these bioconjugates in SK-BR-3 and SKOV-3 cell lines. These targeted, drug encapsulated NPAffibody bioconjugates may be efficacious in treating HER-2 expressing carcinoma

ChemoRad nanoparticles: a novel multifunctional nanoparticle platform for targeted delivery of concurrent chemoradiation

Aim: The development of chemoradiation – the concurrent administration of chemotherapy and radiotherapy – has led to significant improvements in local tumor control and survival. However, it is limited by its high toxicity. In this study, we report the development of a novel NP (nanoparticle) therapeutic, ChemoRad NP, which can deliver biologically targeted chemoradiation. Method: A biodegradable and biocompatible lipid–polymer hybrid NP that is capable of delivering both chemotherapy and radiotherapy was formulated. Results: Using docetaxel, indium111 and yttrium90 as model drugs, we demonstrated that the ChemoRad NP can encapsulate chemotherapeutics (up to 9% of NP weight) and radiotherapeutics (100 mCi of radioisotope per gram of NP) efficiently and deliver both effectively. Using prostate cancer as a disease model, we demonstrated the targeted delivery of ChemoRad NPs and the higher therapeutic efficacy of ChemoRad NPs. Conclusion: We believe that the ChemoRad NP represents a new class of therapeutics that holds great potential to improve cancer treatment

Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release

Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-α and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required

Comparative analysis of unilateral removable partial denture and classical removable partial denture by using finite element method

Introduction. Various mobile devices are used in the therapy of unilateral free-end saddle. Unilateral dentures with precise connectivity elements are not used frequently. In this paper the problem of applying and functionality of unilateral freeend saddle denture without major connector was taken into consideration. Objective. The aim was to analyze and compare a unilateral RPD (removable partial denture) and a classical RPD by calculating and analyzing stresses under different loads. Methods. 3D models of unilateral removable partial denture and classical removable partial denture with casted clasps were made by using computer program CATIA V5 (abutment teeth, canine and first premolar, with crowns and abutment tissues were also made). The models were built in full-scale. Stress analyses for both models were performed by applying a force of 300 N on the second premolar, a force of 500 N on the first molar and a force of 700 N on the second molar. Results. The Fault Model Extractor (FME) analysis and calculation showed the complete behavior of unilateral removable partial denture and abutments (canine and first premolar), as well as the behavior of RPD under identical loading conditions. Applied forces with extreme values caused high stress levels on both models and their abutments within physiological limits. Conclusion. Having analyzed stresses under same conditions, we concluded that the unilateral RPD and classical RPD have similar physiological values.Uvod. U lečenju jednostrane krezubosti koriste se različiti oblici mobilnih nadoknada. Jednostrane kompleksne proteze sa preciznim veznim elementima nisu često u upotrebi. Radi rasvetljenja problema jednostrano slobodnog sedla i primene proteze koja ne poseduje veliku spojnicu, u ovom radu je prikazano istraživanje funkcionalnosti ove manje poznate nadoknade. Cilj rada. Cilj rada je bio da se ispitaju i uporede dobijeni naponi jednostrane kompleksne i konvencionalne parcijalne skeletirane proteze pod opterećenjem. Metode rada. U kompjuterskom programu CATIA V5 napravljeni su trodimenzionalni modeli jednostrane kompleksne proteze s odgovarajućim potpornim strukturama (retencioni zubi - očnjak i prvi premolar s namenskim krunicama, alveole i parodontalni prostor) i klasične skeletirane proteze s livenim kukicama. Radi validnosti dobijenih rezultata, modeliranje je obavljeno u prirodnoj veličini. Primenom metode konačnih elemenata izvršena je komparativna analiza vrednosti dobijenih napona pri opterećenju silama od 300 N u predelu drugog premolara, 500 N u predelu prvog molara i 700 N u predelu drugog molara. Rezultati. Proračun je dao sliku ponašanja celog modela jednostrane kompleksne parcijalne proteze s retencionim zubima i sliku celog modela klasične skeletirane parcijalne proteze koje su predstavljene u obliku napona pri različitim uslovima opterećenja. Uneta opterećenja izazvala su visok nivo napona na modelu i zubima nosačima, ali u granicama fiziološke podnošljivosti. Zaključak. Naponi na potpornim strukturama jednostrane kompleksne proteze koji su nastali usled dejstva primenjenih sila bili su u podnošljivim fiziološkim granicama. U uslovima istog opterećenja primenom metode konačnih elemenata dobijaju se veoma slične vrednosti napona jednostrane kompleksne proteze i konvencionalne skeletirane proteze

IMPORTANCE OF ACUTE PHASE INFLAMMATION SERUM LEVEL MARKERS FOR EARLY DETECTION, FOLOW-UP AND INITIAL PROGNOSIS OF BACTERIAL LOW RESPIRATORY TRACT INFECTIONS IN PATIENTS WITH ALCOHOL LIVER CIRRHOSIS

Combined metabolic and haemodynamic changes in patients (pts) with alcohol liver cirrhosis induce a significant loss of immune response, which represents the main cause of bacterial infections, primarily of low respiratory tract, with high risk of mortality.Considering the influence of bacterial low respiratory tract infections on the course and prognosis of liver cirrhosis, comparing the level of general inflammation response, the clinical data of 67 alcohol liver cirrhosis pts in Child B stage of disease were retrospectively analyzed, diagnosed and treated from September 2001 till February 2006. Regarding the presence of infection, pts were divided in two groups: I-experimental including 37 pts and II-control group with 30 pts.In I group of pts, a significant initial increase of C-reactive protein and fibrinogen serum level (p<0.001) were reported as well as increased erythrocyte sedimentation rate (p<0.05) compared to the control group. The same values were significantly decreased after antibiotic treatment. Gram-negative bacteria were dominant in the culture isolates, the total proteins and albumins serum levels were initially significantly lower (p<0.05), while alanin-aminotranspherasis and lactate dehydrogenase were increased (p<0.05) compared to the control group, with further normalizing tendency at the end of antibiotic treatment.Early detection of bacterial low respiratory tract infections in patients with alcohol liver cirrhosis, by determination of acute phase inflammation serum level markers, is important in achieving the effective and prolonged remission of disease, but it still remains the missing link in complex chains of the unfavorable disease course activation