MMPs-responsive silk spheres for controlled drug release within tumor microenvironment.

Silk is a biocompatible and biodegradable material that enables the formation of various morphological forms, including nanospheres. The functionalization of bioengineered silk makes it possible to produce particles with specific properties. In addition to tumor cells, the tumor microenvironment (TME) includes stromal, immune, endothelial cells, signaling molecules, and the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are overexpressed in TME. We investigated bioengineered spider silks functionalized with MMP-responsive peptides to obtain targeted drug release from spheres within TME. Soluble silks MS12.2MS1, MS12.9MS1, and MS22.9MS2 and the corresponding silk spheres carrying MMP-2 or MMP-2/9 responsive peptides were produced, loaded with doxorubicin (Dox), and analyzed for their susceptibility to MMP-2/9 digestion. Although all variants of functionalized silks and spheres were specifically degraded by MMP-2/9, the MS22.9MS2 nanospheres showed the highest levels of degradation and release of Dox after enzyme treatment. Moreover, functionalized spheres were degraded in the presence of cancer cells releasing MMP-2/9. In the 2D and 3D spheroid cancer models, the MMP-2/9-responsive substrate was degraded and released from spheres when loaded into MS22.9MS2 particles but not into the control MS2 spheres. The present study demonstrated that a silk-based MMP-responsive delivery system could be used for controlled drug release within the tumor microenvironment.

Endotoxin reduction from biotech silk material inhibits the production of anti-silk antibodies in mice.

Eliminating endotoxins is a common problem in the development of biotechnologically produced pharmaceuticals or biomaterials. Residual endotoxins in the final sample may hamper the properties of the product or induce severe adverse effects. Developing an effective downstream purification protocol that ensures a lack of minimal endotoxin content in the final product can be a challenging task. In our previous studies, we developed nanospheres produced from bioengineered silks. Despite their good overall biocompatibility, in vivo characterization of spheres showed mild activation of the immune system (mainly in terms of anti-silk antibody production). Herein, we examined, if the endotoxins delivered with the silk spheres might have contributed to activating the adaptive immune response. We investigated various commercially available methods for endotoxin removal that can be applied as an extra step in downstream endotoxin removal from MS1-type silk proteins. We selected a method that allowed for a 10-fold reduction of endotoxin content in soluble silk and 2-fold in the final product (silk spheres). The reduced level of endotoxins improved the biocompatibility of the silk spheres as these particles induced negligible titers of anti-silk antibodies in an in vivo immune study. Since endotoxins can enhance life-threatening immune responses, it is crucial to optimize the method of their removal before clinical use not only of silk-based products but also of other biomolecules produced biotechnologically.

In vivo study of the immune response to bioengineered spider silk spheres.

Bioengineered MS1 silk is derived from major ampullate spidroin 1 (MaSp1) from the spider Nephila clavipes. The MS1 silk was functionalized with the H2.1 peptide to target Her2-overexpressing cancer cells. The immunogenic potential of drug carriers made from MS1-type silks was investigated. The silk spheres were administered to healthy mice, and then (i) the phenotypes of the immune cells that infiltrated the Matrigel plugs containing spheres (implanted subcutaneously), (ii) the presence of silk-specific antibodies (after two intravenous injections of the spheres), (iii) the splenocyte phenotypes and their activity after restimulation ex vivo in terms of proliferation and cytokine secretion (after single intravenous injection of the spheres) were analyzed. Although the immunogenicity of MS1 particles was minor, the H2.1MS1 spheres attracted higher levels of B lymphocytes, induced a higher anti-silk antibody titer, and, after ex vivo restimulation, caused the activation of splenocytes to proliferate and express more IFN-γ and IL-10 compared with the PBS and MS1 groups. Although the H2.1MS1 spheres triggered a certain degree of an immunological response, multiple injections (up to six times) neither hampered the carrier-dependent specific drug delivery nor induced toxicity, as previously indicated in a mouse breast cancer model. Both findings indicate that a drug delivery system based on MS1-type silk has great potential for the treatment of cancer and other conditions.

Systemic and Local Silk-Based Drug Delivery Systems for Cancer Therapy.

For years, surgery, radiotherapy, and chemotherapy have been the gold standards to treat cancer, although continuing research has sought a more effective approach. While advances can be seen in the development of anticancer drugs, the tools that can improve their delivery remain a challenge. As anticancer drugs can affect the entire body, the control of their distribution is desirable to prevent systemic toxicity. The application of a suitable drug delivery platform may resolve this problem. Among other materials, silks offer many advantageous properties, including biodegradability, biocompatibility, and the possibility of obtaining a variety of morphological structures. These characteristics allow the exploration of silk for biomedical applications and as a platform for drug delivery. We have reviewed silk structures that can be used for local and systemic drug delivery for use in cancer therapy. After a short description of the most studied silks, we discuss the advantages of using silk for drug delivery. The tables summarize the descriptions of silk structures for the local and systemic transport of anticancer drugs. The most popular techniques for silk particle preparation are presented. Further prospects for using silk as a drug carrier are considered. The application of various silk biomaterials can improve cancer treatment by the controllable delivery of chemotherapeutics, immunotherapeutics, photosensitizers, hormones, nucleotherapeutics, targeted therapeutics (e.g., kinase inhibitors), and inorganic nanoparticles, among others.

MS1-type bioengineered spider silk nanoparticles do not exhibit toxicity in an in vivo mouse model.

Background: Due to factors such as silk sequence, purification, degradation, morphology and functionalization, each silk variant should be individually tested for potential toxicity. Aim:  In vivo toxicological evaluation of the previously characterized bioengineered H2.1MS1 spider silk particles that can deliver chemotherapeutics to human epidermal growth factor receptor 2-positive breast cancer. Materials & methods: Silk nanoparticles (H2.1MS1 and control MS1) were administered intravenously to mice, and then the organismal response was assessed. Several parameters of acute and subchronic toxicity were analyzed, including animal mortality and behavior, nanosphere biodistribution, and histopathological analysis of internal organs. Also, the complete blood count, as well as the concentration of biochemical parameters and cytokines in the serum, were examined. Results & conclusion: No toxicity of the systemically administrated silk nanosphere was observed, indicating their potential application in biomedicine.

Functionalized silk spheres selectively and effectively deliver a cytotoxic drug to targeted cancer cells in vivo.

BACKGROUND: Chemotherapy is often a first-line therapeutic approach for the treatment of a wide variety of cancers. Targeted drug delivery systems (DDSs) can potentially resolve the problem of chemotherapeutic drug off-targeting effects. Herein, we examined in vivo models to determine the efficacy of Her2-targeting silk spheres (H2.1MS1) as DDSs for delivering doxorubicin (Dox) to Her2-positive and Her2-negative primary and metastatic mouse breast cancers. RESULTS: The specific accumulation of H2.1MS1 spheres was demonstrated at the site of Her2-positive cancer. Dox delivered only by functionalized H2.1MS1 particles selectively inhibited Her2-positive cancer growth in primary and metastatic models. Moreover, the significant effect of the Dox dose and the frequency of treatment administration on the therapeutic efficacy was indicated. Although the control MS1 spheres accumulated in the lungs in Her2-positive metastatic breast cancer, the Dox-loaded MS1 particles did not treat cancer. Histopathological examination revealed no systemic toxicity after multiple administrations and at increased doses of Dox-loaded silk spheres. Although the studies were performed in immunocompetent mice, the H2.1MS1 silk spheres efficiently delivered the drug, which exerted a therapeutic effect. CONCLUSION: Our results indicated that functionalized silk spheres that enable cell-specific recognition, cellular internalization, and drug release represent an efficient strategy for cancer treatment in vivo.

Silk Particles as Carriers of Therapeutic Molecules for Cancer Treatment.

Although progress is observed in cancer treatment, this disease continues to be the second leading cause of death worldwide. The current understanding of cancer indicates that treating cancer should not be limited to killing cancer cells alone, but that the target is the complex tumor microenvironment (TME). The application of nanoparticle-based drug delivery systems (DDS) can not only target cancer cells and TME, but also simultaneously resolve the severe side effects of various cancer treatment approaches, leading to more effective, precise, and less invasive therapy. Nanoparticles based on proteins derived from silkworms' cocoons (like silk fibroin and sericins) and silk proteins from spiders (spidroins) are intensively explored not only in the oncology field. This natural-derived material offer biocompatibility, biodegradability, and simplicity of preparation methods. The protein-based material can be tailored for size, stability, drug loading/release kinetics, and functionalized with targeting ligands. This review summarizes the current status of drug delivery systems' development based on proteins derived from silk fibroin, sericins, and spidroins, which application is focused on systemic cancer treatment. The nanoparticles that deliver chemotherapeutics, nucleic acid-based therapeutics, natural-derived agents, therapeutic proteins or peptides, inorganic compounds, as well as photosensitive molecules, are introduced.

Drug affinity and targeted delivery: double functionalization of silk spheres for controlled doxorubicin delivery into Her2-positive cancer cells.

BACKGROUND: The optimal drug delivery system should be biocompatible, biodegradable, and allow the sustained release of the drug only after it reaches the target cells. Silk, as a natural polymer, is a great candidate for building drug carriers. Genetically engineered silks offer the possibility of functionalization. Previously, we characterized bioengineered silk spheres that were functionalized with H2.1 peptide that selectively delivered a drug to Her2-positive cancer cells. However, drug leakage from the silk spheres showed the need for improved control. RESULTS: To control the drug loading and release, we designed and produced functional silk (DOXMS2) that contains a DOX peptide with an affinity for doxorubicin. The DOXMS2 spheres showed the decreased release of doxorubicin compared with MS2 particles. Next, the DOXMS2 silk was blended with the H2.1MS1 polymer to improve the control of doxorubicin binding and release into Her2-positive cancer cells. The H2.1MS1:DOXMS2 particles showed the highest doxorubicin-loading capacity and binding per cell, which resulted in the highest cytotoxic effect compared with that of other sphere variants. Since drug release at a pH of 7.4 from the blended H2.1MS1:DOXMS2 particles was significantly lower than from blended spheres without DOXMS2 silk, this indicated that such particles could control the release of the drug into the circulatory system before the carrier reached the tumor site. CONCLUSIONS: This strategy, which is based on the blending of silks, allows for the generation of particles that deliver drugs in a controlled manner.

Silk Materials Functionalized via Genetic Engineering for Biomedical Applications.

The great mechanical properties, biocompatibility and biodegradability of silk-based materials make them applicable to the biomedical field. Genetic engineering enables the construction of synthetic equivalents of natural silks. Knowledge about the relationship between the structure and function of silk proteins enables the design of bioengineered silks that can serve as the foundation of new biomaterials. Furthermore, in order to better address the needs of modern biomedicine, genetic engineering can be used to obtain silk-based materials with new functionalities. Sequences encoding new peptides or domains can be added to the sequences encoding the silk proteins. The expression of one cDNA fragment indicates that each silk molecule is related to a functional fragment. This review summarizes the proposed genetic functionalization of silk-based materials that can be potentially useful for biomedical applications.

Brain perfusion evaluated by perfusion-weighted magnetic resonance imaging before and after stenting internal carotid artery stenosis in asymptomatic and symptomatic patients.

PURPOSE: To evaluate the brain perfusion with MRI perfusion weighted imaging (PWI) before and after ICA stenting in asymptomatic and symptomatic patients. MATERIALS AND METHODS: PWI was performed 3-21 days before and 3 days after ICA stenting in 31 asymptomatic patients with ICA >70% stenosis - Group I, and in 14 symptomatic patients with ICA >50% stenosis - Group II. PWI was evaluated qualitatively and quantitatively in 5 cerebral territories with: mean transit time (MTT), cerebral blood volume (CBV) and cerebral blood flow (CBF). Mean values of perfusion parameters were measured before and after stenting ΔMTT, ΔCBV, ΔCBF were calculated as subtraction of after-treatment values from those before treatment. RESULTS: In qualitative evaluation after ICA stenting perfusion was normalized in 21 patients (80.8%) in Group I and in 8 patients (80%) in Group II. In quantitative estimation MTT decreased significantly after CAS on stented side vs. non-stented side in all examined patients regardless of the group, p<0.05. MTT decreased more in Group II than in Group I in all territories (p<0.05) with the exception of temporal lobe. CBV and CBF have shown insignificant differences.

[Current status of drug-eluting stents and drug-eluting balloons in patients with stable coronary artery disease--an expert consensus document of the Association for Percutaneous Cardiovascular Interventions and Polish Cardiac Society]. / Stanowisko grupy ekspertów dotyczace zastosowaniastentów oraz balonów uwalniajacych leki antyproliferacyjneu pacjentów z choroba wiencowa przyjete przez Asocjacje Interwencji Sercowo−Naczyniowychi Polskie Towarzystwo Kardiologiczne.

Despite a dramatic reduction in restenosis and target vessel revascularisation rates by drug eluting stents (DES), conflicting concerns have been raised over the risk for late DES thrombosis when compared to bare metal stents. Certainly, the heterogeneity of DES results from the introduction of a great variety of new DES types with diverse efficacy and safety parameters. Furthermore, we observe a steady increase in DES availability without parallel and robust data from randomised clinical trials. Thus, the postulated class effect of DES should be regarded as non-obligatory. This article is based on 110 randomised DES trials performed on 72 305 patients. Current status of DES and guidelines for DES implantation in various clinical scenarios were discussed.

Association of non-specific inflammatory activation with early mortality in patients with ST-elevation acute coronary syndrome treated with primary angioplasty.

BACKGROUND: The association of inflammatory markers with mortality in ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI) remains controversial, so in the present study the relationships of high-sensitivity C-reactive protein (hs-CRP), total white blood cell (WBC) count, neutrophil (N) and lymphocyte (L) counts and the N/L ratio with occurrence of in-hospital mortality were assessed in patients with STEMI treated with primary PCI. METHODS AND RESULTS: Inflammatory parameters were assessed on admission in 1,078 consecutive, unselected patients with STEMI admitted for primary PCI. In-hospital death occurred in 6.3% of the patients. Of the inflammatory parameters, only hs-CRP (p<0.001), and the WBC (p=0.004) and N (p=0.020) counts were predictors of death in the univariate analyses. After adjustment for other baseline clinical variables both hs-CRP and WBC count retained their independent association with mortality when analyzed both in 2 separate and in 1 multivariable models. CONCLUSIONS: Both hs-CRP and the WBC count may independently of each other predict early outcomes in STEMI patients treated with primary PCI, which suggests different pathological significance of these 2 non-specific inflammatory markers in STEMI.

Differences in presentation and management of stable angina from East to West in Europe: a comparison between Poland and the UK.

AIMS: Variations in the resources, stability and priorities of health care systems conceivably affect their capacity to implement health care reform and ensure an evidence based approach to health care. Such variation may partially account for differences in cardiovascular mortality rates between former communist states in Central Europe and Western European countries, but specific data on this subject is sparse. The aim of this study was to compare the presentation of stable angina to cardiology services in Poland vs. the United Kingdom, the management of the condition in relation to existing European guidelines and clinical outcome. METHODS AND RESULTS: Data was collected as part of a prospective observational cohort study of stable angina in Europe. Information was recorded on referral patterns, clinical presentation and the use of pharmacological therapies, investigations, revascularisation and cardiovascular events during 1 year of follow up. A total of 571 patients with stable angina were enrolled in Poland and 319 in the UK. Patients presenting to cardiology services in Poland were less likely to be referred by a primary care physician, younger, and had more adverse clinical risk predictors at presentation. Non-invasive investigation and coronary angiography were performed less frequently in Poland, but waiting times for invasive assessment were shorter. European guidelines with regard to the use of evidence based secondary preventative medical therapy were applied widely by cardiologists in both countries. No differences were observed in rates of cardiovascular events. CONCLUSIONS: The use of evidence based pharmacological therapy was equally high in both countries, but guidelines regarding investigation were less completely adhered to in Poland, where invasive assessment and subsequent management was prompt but only performed in a highly selected proportion of the population with stable angina.

Comparison of different methods of ST segment resolution analysis for prediction of 1-year mortality after primary angioplasty for acute myocardial infarction.

BACKGROUND: Resolution of ST segment elevation corresponds with myocardial tissue reperfusion and correlates with clinical outcome after ST elevation myocardial infarction. Simpler method evaluating the extent of maximal deviation persisting in a single ECG lead was an even stronger mortality predictor. Our aim was to evaluate and compare prognostic accuracy of different methods of ST segment elevation resolution analysis after primary percutaneous coronary intervention (PCI) in a real-life setting. METHODS: Paired 12-lead ECGs were analyzed in 324 consecutive and unselected patients treated routinely with primary PCI in a single high-volume center. ST segment resolution was quantified and categorized into complete, partial, or none, upon the (1) sum of multilead ST elevations (sumSTE) and (2) sum of ST elevations plus reciprocal depressions (sumSTE+D); or into the low-, medium-, and high-risk groups by (3) the single-lead extent of maximal postprocedural ST deviation (maxSTE). RESULTS: Complete, partial, and nonresolution groups by sumSTE constituted 39%, 40%, and 21% of patients, respective groups by sumSTE+D comprised 40%, 39%, and 21%. The low-, medium-, and high-risk groups constituted 43%, 32%, and 25%. One-year mortality rates for rising risk groups by sumSTE were 4.7%, 10.2%, and 14.5% (P = 0.049), for sumSTE+D 3.8%, 9.6%, and 17.6% (P = 0.004) and for maxSTE 5.1%, 6.7%, and 18.5% (P = 0.001), respectively. After adjustment for multiple covariates only maxSTE (high vs low-risk, odds ratio [OR] 3.10; 95% confidence interval [CI] 1.11-8.63; P = 0.030) and age (OR 1.07; 95% CI 1.02-1.11; P = 0.002) remained independent predictors of mortality. CONCLUSIONS: In unselected population risk stratifications based on the postprocedural ST resolution analysis correlate with 1-year mortality after primary PCI. However, only the single-lead ST deviation analysis allows an independent mortality prediction.

Comparison of prognostic value of epicardial blood flow and early ST-segment resolution after primary coronary angioplasty. ANIN--Myocardial Infarction Registry.

BACKGROUND: TIMI scale is commonly used for angiographic assessment of reperfusion effectiveness and early risk stratification in patients treated with primary angioplasty for ST-elevation myocardial infarction (STEMI). Since ST-resolution analysis allows a noninvasive insight into the reperfusion status at the myocardial tissue level, it may be a better predictor of outcome after primary angioplasty. AIM: To compare the prognostic value of the reperfusion effectiveness evaluation based on either the epicardial blood flow assessment according to the TIMI scale, or ST-segment resolution analysis in patients treated with primary coronary angioplasty for STEMI. METHODS: 324 consecutive patients treated within 12 hours from the pain onset were studied. Based on the analysis of maximal ST-segment elevation/depression identified in a single ECG lead recorded after the procedure (maxSTE), patients were classified into groups of high versus medium/low risk. Independently, distinguished were groups with restored normal (TIMI 3) and abnormal (TIMI 0-2) final blood flow in infarct related artery. RESULTS: The 30-day and one-year mortality rates were higher in the high-risk maxSTE group (25% of all patients) than in the other patients (14.8% vs. 2.5%, p<0.001 and 18.5% vs. 5.4%, p<0.001 respectively). In subjects (82%) with restored TIMI grade 3 blood flow, mortality at one-month and one-year was lower than in the group with abnormal final blood flow (3.1% vs. 15.6%, p=0.001 and 6.2% vs. 18.8%, p=0.005). Comparison in multivariate analysis revealed that maxSTE stratification but not final TIMI grade assessment remained an independent predictor of both, 30-day and one-year mortality (high vs. medium/low-risk category; OR 5.3, 95% CI 1.6-16.7, p=0.005, and OR 3.3, 95% CI 1.4-7.8, p=0.007, respectively). Furthermore, maxSTE proved to stratify the risk of death even in subgroup of patients with restored normal blood flow (OR 6.2, 95% CI 1.4-27.8, p=0.016, and OR 3.0, 95% CI 1.1-8.7, p=0.039, respectively). CONCLUSIONS: Analysis of extent of maximal ST-segment elevation or depression identified in a single ECG lead after primary coronary angioplasty allows better prognosis of subsequent 30-day and one-year mortality than the assessment of final epicardial blood flow, stratifying risk of death even in a subgroup of patients with restored normal blood flow.

Cardiac amyloidosis diagnosed by endomyocardial biopsy. Clinical, histopathological, immunohistochemical and ultrastructural studies.

BACKGROUND: The heart is often involved by primary (AL) and familial transthyretin-related (ATTR) amyloidosis. Endomyocardial biopsy is a valuable diagnostic method, useful in detection and recognition of the type of amyloid. AIM: The aim of the study was to determine the type of amyloid deposits found in endomyocardial biopsies, using histochemical, immunohistochemical and ultrastructural methods. The correlations between morphological and clinical parameters were evaluated. METHODS AND RESULTS: During 1999-2004, endomyocardial biopsy was performed in 41 patients hospitalized in our institution due to heart failure of unknown etiology. Amyloid deposits were revealed in 3 patients, in 2 of them the intraoperative specimens were also taken. The deposits were located in myocardial interstitium as well as in the wall of small vessels. One patient showed deposits in the atrial and pericardial walls. Electron microscopy study revealed amyloid fibers adhering to the basement membranes of the myocardial cells and small vessels. Immunohistochemical staining determined two types of amyloid. A woman aged 42 showed transthyretin-related amyloidosis, most probably hereditary. A woman aged 69 exhibited the light chains amyloidosis (AL-lambda). In contrast, diagnosis was not certain (most probably ATTR) in a 35 year old woman. Essential involvement of the pericardium was observed in two younger patients. They underwent pericardiotomy. The survival time (from diagnosis to death) was one year in the patient with amyloidosis ATTR, 17 days in the patient with the AL type, and 2 years in the patient with amyloidosis of undetermined type. CONCLUSIONS: Cardiac amyloidosis is associated with poor prognosis. Better recognition of this disease may allow the early diagnosis and institution of modern therapy.

Acute myocardial infarction complicated by cardiogenic shock. In-hospital and mid-term results of invasive treatment in the National Institute of Cardiology, Warsaw-Anin.

BACKGROUND: Mortality in acute myocardial infarction (MI) complicated by cardiogenic shock approaches 90%, regardless of the type of pharmacological treatment. AIM: To assess in-hospital and mid-term results of invasive treatment of patients with acute MI with ST segment elevation (STEMI) complicated by cardiogenic shock. METHODS: From a prospective registry of all patients admitted to our institution for urgent coronary angiography due to acute coronary syndrome between February 2001 and June 2002, patients with STEMI, symptom duration up to 12 hours and cardiogenic shock diagnosed on admission were identified. The in-hospital and mid-term outcome of 37 patients (mean age 65 years, range 54-77, 68% of males) treated with primary percutaneous coronary intervention (PCI) was analysed. RESULTS: Of the 41 patients with STEMI and cardiogenic shock, total occlusion or critical stenosis of a coronary artery were found in 38 patients. One patient with the occlusion of three main coronary arteries underwent urgent surgical revascularisation and remains alive after an 18-month follow-up. In the remaining 37 patients primary PCI of an infarct-related artery was performed (stent implantation in 70%, abciximab administration in 54%) which restored normal blood flow (TIMI grade 3 flow) in 54% of subjects. In patients with TIMI grade 3 flow the in-hospital mortality was 25%. Of the whole PCI-treated group, 18 (48.6%) patients died during stay in our institution, an additional two - after transfer to another hospital, and one - during a 19-month follow-up period. The remaining 16 patients remain alive (median follow-up of 8 months). CONCLUSIONS: Invasive treatment of patients with STEMI complicated by cardiogenic shock significantly reduces mortality in this high-risk population. The mid-term results in patients discharged from hospital are good. Invasive treatment of acute MI should be accessible for all patients with extensive acute MI.