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Covid-19 Impact On Global Nanotechnology Drug Delivery Market : Industry Analysis and Forecast (2019-2026): By Technology, Application and Region. -…
Global Nanotechnology Drug Delivery Market was valued US$ XX Bn in 2018 and is expected to reach US$ 98.2 Bn by 2026, at a XX% CAGR of around during a forecast period.
Various novel technologies for developing effective drug delivery systems came into existence among which nanotechnology platforms for achieving targeted drug delivery are gaining prominence nowadays. Research in the medical field includes the development of drug nanoparticles, polymeric and inorganic biodegradable nano-carriers for drug delivery, and surface engineering of carrier molecules.
The report contains a detailed list of factors that will drive and restrain the growth of the Nanotechnology Drug Delivery Market. Such as, rapidly expanding areas of research and development to develop novel nano-medicine are expected to drive the nanotechnology drug delivery market growth in the future. Additionally, one of the major factors assisting market growth is the growing prevalence of infectious diseases and cancer, developing nanotechnology research, and increasing demand for novel drug delivery systems. However, high cost coupled with stringent regulatory scenario hinders the market growth to some extent.
Nanoparticles are expected to account for the largest XX% market share by 2026. The segment dominated the market as key nanoparticles like gold nanoparticles, dendrimers, and fullerenes are used in pharmaceutical drug delivery.The report offers a brief analysis of the major regions in the global nanotechnology drug delivery market, namely, APAC, Europe, North America, South America, and the Middle East & Africa. North America dominated the nanotechnology drug delivery market in 2018, because of high medical reimbursement facilities, and technological advancement. The APAC is projected to have the fastest growth, owing to a rapidly increasing population, an increase in consumer awareness, favorable government policies, modernization of healthcare infrastructure, and growing medical tourism industry in developing economies such as China, and India in this region.
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Nanotechnology drug delivery market report gives a competitive analysis of the individual standing of the companies against the global landscape of the medical industry. The forecast also provides the estimated trends in demand for the global market and their impact on the sizes of these companies to help the reader curate profitable business strategies. Such as Pfizer, Inc., AstraZeneca and Amgen signed agreements to collaborate with BIND Therapeutics to develop nano-medicines. These initiatives are expected to fuel the growth of the nanotechnology drug delivery market in the upcoming future.
The objective of the report is to present comprehensive analysis of Global Nanotechnology Drug Delivery Market including all the stakeholders of the industry. The past and current status of the industry with forecasted market size and trends are presented in the report with the analysis of complicated data in simple language. The report covers the all the aspects of industry with dedicated study of key players that includes market leaders, followers and new entrants by region. PORTER, SVOR, PESTEL analysis with the potential impact of micro-economic factors by region on the market have been presented in the report. External as well as internal factors that are supposed to affect the business positively or negatively have been analyzed, which will give clear futuristic view of the industry to the decision makers.
The report also helps in understanding Global Nanotechnology Drug Delivery Market dynamics, structure by analyzing the market segments, and project the Global Nanotechnology Drug Delivery Market size. Clear representation of competitive analysis of key players by type, price, financial position, product portfolio, growth strategies, and regional presence in the Global Nanotechnology Drug Delivery Market make the report investors guide.
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The report study has analyzed revenue impact of covid-19 pandemic on the sales revenue of market leaders, market followers and disrupters in the report and same is reflected in our analysis.Scope of the Global Nanotechnology Drug Delivery Market
Global Nanotechnology Drug Delivery Market, by Technology
Nanocrystals Nanoparticleso Dendrimerso Gold Nanoparticleso Dendrimerso Fullereneso Others Liposomes Micelles Nanotubes OthersGlobal Nanotechnology Drug Delivery Market, by Application
Neurology Oncology Cardiovascular/Physiology Anti-inflammatory/Immunology Anti-infective OthersGlobal Nanotechnology Drug Delivery Market, by Region
North America Asia Pacific Europe Middle East & Africa South AmericaKey players operating in the Global Nanotechnology Drug Delivery Market
Johnson & Johnson Merck & Co Roche Bayer Novartis Pharmaceuticals Pfizer AstraZeneca Amgen Celgene Corporation Angiotech Pharmaceuticals Capsulution Pharma AlphaRx Inc. Calando Pharmaceuticals Copernicus Therapeutics Elan Corporation Nanotherapeutics PAR Pharmaceutica Taiwan Liposome Co. AbbVie, Inc
Major Table of Contents Report
Chapter One: Nanotechnology Drug Delivery Market Overview
Chapter Two: Manufacturers Profiles
Chapter Three: Global Nanotechnology Drug Delivery Market Competition, by Players
Chapter Four: Global Nanotechnology Drug Delivery Market Size by Regions
Chapter Five: North America Nanotechnology Drug Delivery Revenue by Countries
Chapter Six: Europe Nanotechnology Drug Delivery Revenue by Countries
Chapter Seven: Asia-Pacific Nanotechnology Drug Delivery Revenue by Countries
Chapter Eight: South America Nanotechnology Drug Delivery Revenue by Countries
Chapter Nine: Middle East and Africa Revenue Nanotechnology Drug Delivery by Countries
Chapter Ten: Global Nanotechnology Drug Delivery Market Segment by Type
Chapter Eleven: Global Nanotechnology Drug Delivery Market Segment by Application
Chapter Twelve: Global Nanotechnology Drug Delivery Market Size Forecast (2019-2026)
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Global Nano-SiO2 Market 2020 Impact of COVID-19, Future Growth Analysis and Challenges | PPG, Akzonobel, Sherwin-Williams, RPM International, Axalta…
The Global Nano-SiO2 Market report focuses on market size, status and forecast 2020-2027, along with this, report also focuses on market opportunities and treats, risk analysis, strategic and tactical decision-making and evaluating the market. The Nano-SiO2 market report provides data and information on changing investment structure, technological advancements, market trends and developments, capacities, and detail information about the key players of the global Maarket_Keyword market. In addition to this, report also involves development of the Nano-SiO2 market in major region across the world.
Key Players for Global Nano-SiO2 Market:
The global Nano-SiO2 market report profiles major key players of the market on the basis of business strategies, financial weaknesses and strengths and recent development.
PPGAkzonobelSherwin-WilliamsRPM InternationalAxaltaBASFKansai PaintNanomechEIKOSTelsa Nano CoatingsInframat CorporationNanophaseDiamon-Fusion InternationalNanovere Technologies
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The Nano-SiO2 market report also states demand and supply figures, revenue, production, import/export consumption as well as future strategies, sales volume, gross margins, technological developments, cost and growth rate. The Global Nano-SiO2 Market report also delivers historical data from 2015 to 2020 and forecasted data from 2020 to 2027, along with SWOT analysis data of the market. This report includes information by types, by application, by region and by manufacturers or producers.
The recent outburst of the COVID-19 (Corona Virus Disease) has led the global Nano-SiO2 market to render new solutions for combatting with the rising demand for protection against the virus. Due to this outbreak, remote patient monitoring, inpatient monitoring and interactive medicine is expected to gain grip at this time.
Global Nano-SiO2 Market: Segmentation
Global Nano-SiO2 Market Segmentation: By Types
Dry MethodWet Method
Global Nano-SiO2 Market segmentation: By Applications
Global Nano-SiO2 Market Segmentation: By Region
Global Nano-SiO2 market report categorized the information and data according to the major geographical regions like,
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The Global Nano-SiO2 market is displayed in 13 Chapters:
Chapter 1: Market Overview, Drivers, Restraints and OpportunitiesChapter 2: Market Competition by ManufacturersChapter 3: Production by RegionsChapter 4: Consumption by RegionsChapter 5: Production, By Types, Revenue and Market share by TypesChapter 6: Consumption, By Applications, Market share (%) and Growth Rate by ApplicationsChapter 7: Complete profiling and analysis of ManufacturersChapter 8: Manufacturing cost analysis, Raw materials analysis, Region-wise manufacturing expensesChapter 9: Industrial Chain, Sourcing Strategy and Downstream BuyersChapter 10: Marketing Strategy Analysis, Distributors/TradersChapter 11: Market Effect Factors AnalysisChapter 12: Market ForecastChapter 13: Nano-SiO2 Research Findings and Conclusion, Appendix, methodology and data source
Global Nanomedicine Market SHARE, SIZE 2020| EMERGING RAPIDLY WITH LATEST TRENDS, GROWTH, REVENUE, DEMAND AND FORECAST TO 2026 – The Collegian
The recently published research report entitled Global Nanomedicine Market sheds light on critical aspects of the market like market size estimations, company and market best practices, market dynamics, market segmentation, competitive landscaping and benchmarking, opportunity analysis, economic forecasting, industry-specific technology solutions, guideline analysis, and in-depth benchmarking of vendor offerings. The report provides a clear understanding of the current and future scenarios and trends of the global Nanomedicine market. The report tracks an array of important market-related aspects which can be listed as follows; the demand and supply chain, the competitive landscape, leading industries shares, profit margin, and profiles of leading companies of the global market.
This report takes into account the current and future impacts of COVID-19 on this industry and offers you an in-depth analysis of Global Trans Resveratrol Market.
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The section offers great insights such as market revenue and market share of the global Nanomedicine market. The report explains a competitive edge over players competitors. Leading as well as prominent players of the global market are broadly studied on the basis of key factors. The report offers a comprehensive analysis and accurate statistics on sales by the player for the period 2015-2020. The report includes the forecasts, analysis, and discussion of important industry trends, market size, market share estimates, and profiles of the leading industry players. Company profile section of players such as Combimatrix, Ablynx, Abraxis Bioscience, Celgene, Mallinckrodt, Arrowhead Research, GE Healthcare, Merck, Pfizer, Nanosphere, Epeius Biotechnologies, Cytimmune Sciences, Nanospectra Biosciences,
Product segment analysis:
Application segment analysis: Segmentation encompasses oncology, Infectious diseases, Cardiology, Orthopedics, Other
To comprehend global Nanomedicine market dynamics in the world mainly, the worldwide market is analyzed across major global regions: North America (United States, Canada, Mexico), Asia-Pacific (China, Japan, South Korea, India, Australia, Indonesia, Thailand, Malaysia, Philippines, Vietnam), Europe (Germany, France, UK, Italy, Russia, Rest of Europe), Central & South America (Brazil, Rest of South America), Middle East & Africa (GCC Countries, Turkey, Egypt, South Africa, Rest of Middle East & Africa)
Moreover, the report elaborates different internal and external factors of the global Nanomedicine market. It uses numerous graphical presentation techniques such as graphs, tables, charts, pictures, and flowcharts. The report further focuses on market dynamics, growth drivers, developing market segments, and the market growth curve based on past, present, and future market data. The up-to-date, complete product knowledge, end-users, industry growth will drive profitability and revenue. Various important factors such as market trends, revenue growth patterns market shares, and demand and supply are included in the market research report for every industry.
The Key Highlights of The Report:
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Transient stealth coating of liver sinusoidal wall by anchoring two-armed PEG for retargeting nanomedicines – Science Advances
A major critical issue in systemically administered nanomedicines is nonspecific clearance by the liver sinusoidal endothelium, causing a substantial decrease in the delivery efficiency of nanomedicines into the target tissues. Here, we addressed this issue by in situ stealth coating of liver sinusoids using linear or two-armed poly(ethylene glycol) (PEG)conjugated oligo(l-lysine) (OligoLys). PEG-OligoLys selectively attached to liver sinusoids for PEG coating, leaving the endothelium of other tissues uncoated and, thus, accessible to the nanomedicines. Furthermore, OligoLys having a two-armed PEG configuration was ultimately cleared from sinusoidal walls to the bile, while OligoLys with linear PEG persisted in the sinusoidal walls, possibly causing prolonged disturbance of liver physiological functions. Such transient and selective stealth coating of liver sinusoids by two-arm-PEG-OligoLys was effective in preventing the sinusoidal clearance of nonviral and viral gene vectors, representatives of synthetic and nature-derived nanomedicines, respectively, thereby boosting their gene transfection efficiency in the target tissues.
Nanomedicines have been widely studied for the efficient delivery of therapeutic and diagnostic agents into target tissues (16). However, nanomedicines are exposed to several clearance mechanisms, such as reticuloendothelial system (RES) uptake, after their systemic administration (79). Among these mechanisms, liver sinusoidal endothelial cells (LSECs) express numerous types of scavenger receptors for capturing a variety of nanomedicines and have high endocytic activity to clear them actively from the blood circulation (1012). The targets of LSEC-mediated clearance include both synthetic and nature-derived nanomedicines, such as viral gene vectors (13, 14), limiting their delivery efficiency to the target tissues.
To address this issue of LSEC-mediated clearance, the stealth coating of nanomedicines, e.g., by poly(ethylene glycol) (PEG), which allows nanomedicines to persist in the blood circulation for hours to days, has been widely attempted (1518). However, depending on the formulation of the nanomedicine and its drug contents, it is often difficult to obtain sufficient stealth coating to completely inhibit the clearance mechanisms without compromising nanomedicine functionality (1922). Thus, a combination of other strategies is required. The modulation of host-tissue clearance mechanisms is a promising option. For this purpose, previous studies have attempted to saturate the availability of clearing sites, e.g., by preinjecting scavenger receptor ligands, such as fucoidan (23), polyinosinic acid (poly-I) (24), and dextran sulfate (DS) (25), or decoy nanoparticles, such as polymer-albumin nanoparticles (26) and cationic liposomes (27). However, this strategy has two major problems. First, agents used for receptor saturation inhibit only specific mechanisms of sinusoidal clearance, depending on the receptors or clearance sites that they target, despite the fact that the liver sinusoid has diverse clearance pathways. Even a single nanomedicine can be recognized by several receptors (12, 28, 29), such that the simultaneous inhibition of various clearance mechanisms is preferred. Second, the receptor saturation strategy often raises safety concerns, including inflammatory responses induced by fucoidan (30) or poly-I (31) and anticoagulation associated with the administration of DS (32).
To circumvent these issues, herein we propose transient and selective stealth coating of liver sinusoidal endothelium, using precisely designed PEGylated oligocation (Fig. 1). In contrast to the previous strategy of receptor saturation, PEG coating of liver sinusoidal endothelium would be effective for the simultaneous inhibition of various clearance mechanisms. The coating should be transient and selective to the liver sinusoid to avoid toxicity concerns. This was achieved by using oligo(l-lysine) (OligoLys) conjugated with two-armed PEG at its carboxyl end (two-arm-PEG-OligoLys) for anchoring PEG to liver sinusoidal walls. The PEGylation of OligoLys allowed us to avoid the nonspecific attachment of OligoLys to the extra-liver endothelium, presumably via the steric repulsion of PEG, with preserved binding capability to liver sinusoidal endothelium, which may have high binding affinity to oligocations because of the abundance of heparan sulfate proteoglycans and scavenger receptors (11, 33, 34). The clearance behavior of the PEGylated OligoLys was successfully controlled by optimizing the PEG configuration, with two-arm-PEG-OligoLys showing transient PEG coating to the liver sinusoidal endothelium, followed by gradual biliary clearance, while the OligoLys conjugated with one-armed (linear) PEG (one-arm-PEG-OligoLys) bound to the sinusoidal endothelium persistently. Subsequently, transient and selective stealth coating of liver sinusoids by two-arm-PEG-OligoLys was found to be effective in preventing the sinusoidal clearance of nonviral and viral gene vectors, providing an increased gene transfection efficiency in their target tissues via their relocation from the liver sinusoid to the tissues.
(A) OligoLys with 20 Lys units conjugated with two linear chains of 40-kDa PEG at its carboxyl end (two-arm-PEG-OligoLys). (B) Schematic illustration of in situ stealth coating of liver sinusoidal wall. Two-arm-PEG-OligoLys selectively attaches to the sinusoidal wall to prevent the attachment of nanomedicines, such as polyplex micelle (PM) and adeno-associated virus (AAV), to the wall via stealth property of PEG. Two-arm-PEG-OligoLys is gradually cleared to the bile to avoid prolonged disturbance of liver sinusoid functions.
Short OligoLys with approximately 20 Lys units was used, as the shortening of the oligo-polycation is an effective strategy to circumvent toxicity concerns (35, 36). OligoLys was PEGylated in two different methods, using either one- or two-armed PEG. A single linear chain of 80-kDa PEG or double linear chains of 40-kDa PEG were conjugated to OligoLys at the proximal -NH2 terminus of PEG by forming a stable covalent amide bond to the distal carboxyl end of OligoLys (Fig. 2A). We selected the PEGylated OligoLys samples to have the same total Mw (weight-average molecular weight) of PEG in each molecule, i.e., 80 kDa. Total PEG Mw was set to 80 kDa for avoiding renal clearance of PEGylated OligoLys (37), which may influence its sinusoidal coating behavior. Note that each molecule of two-arm-PEG-OligoLys has two 40-kDa PEG strands, meaning that total Mw of PEG per OligoLys strand in the molecule is set at 80 kDa, and this is the same PEG Mw ratio to OligoLys as that in each of the one-arm-PEG-OligoLys molecule with a single strand of 80-kDa PEG. In this way, we can faithfully evaluate the effect of PEG configuration (linear versus two-arm branched) without an influence of total Mw of PEG fraction in each PEGylated OligoLys molecule. These PEGylated OligoLys formulations were labeled with a single molecule of Alexa Fluor 594 at the OligoLys main chain -NH2 group for the real-time fluorescence observation of their pharmacokinetic behaviors in living mice using intravital confocal laser scanning microscopy (IVCLSM).
(A) Chemical structures of one-arm-PEG-OligoLys (top, left), two-arm-PEG-OligoLys (top, right) with or without Alexa594 labeling (bottom). (B to D) Alexa594-labeled OligoLys with or without PEGylation was intravenously injected. Five minutes or 1 hour after the injection, earlobe dermis was observed using IVCLSM. (B) One-arm-PEG-OligoLys. (C) Two-arm-PEG-OligoLys. (D) Non-PEGylated OligoLys. Arrowheads, capillary walls. Two-way arrows, capillary lumen.
When observing the earlobe dermis, a representative connective tissue, after intravenous injection of one- and two-arm-PEG-OligoLys, the fluorescence intensity of the blood vessel walls was comparable with that of the lumen (Fig. 2, B and C), indicating no PEGylated OligoLys attachment to the vessel walls of the earlobe. On the contrary, non-PEGylated OligoLys with approximately 28 Lys units was aligned to the vessel walls of the earlobe as early as 5 min after injection (Fig. 2D). Thus, the attachment of OligoLys to the vessel walls of a connective tissue was successfully avoided by PEGylation of OligoLys, presumably due to stealth properties of PEG.
In sharp contrast, both one- and two-arm-PEG-OligoLys were attached to the vessel walls of the liver sinusoid within 5 min after injection (Fig. 3, A and B). Quantitative analysis revealed a much higher fluorescence intensity of the sinusoidal wall compared with the lumen (Fig. 3, C and D). This observation indicates the successful PEG coating of the liver sinusoidal wall after the injection of one- and two-arm-PEG-OligoLys. These PEGylated OligoLys formulations attached more efficiently to the blood vessel walls of the liver compared with those of the connective tissue (Fig. 2, B and C). Such selective binding of one- and two-arm-PEG-OligoLys to the liver sinusoidal wall may be attributed to the abundancy of anionic proteoglycans, such as heparan sulfate proteoglycans, present on the sinusoidal extracellular matrix, which can capture oligocations (33, 34), as well as to the high expression levels of scavenger receptors, which recognize cationic macromolecules, on sinusoidal cells (11).
(A to D) IVCLSM images after injection of Alexa594-labeled one-arm-PEG-OligoLys (A) and two-arm-PEG-OligoLys (B). Green, autofluorescence of liver parenchyma. Red, one- and two-arm-PEG-OligoLys. Presumable regions of bile canaliculi are encircled with white dotted lines. Intensity profiles of Alexa594 in the white arrows in (A) and (B) are shown in (C) and (D), respectively. (C) One-arm-PEG-OligoLys. (D) Two-arm-PEG-OligoLys. (E) Bile ducts were visualized using 5-carboxyfluorescein (CF, green). Then, Alexa594-labeled two-arm-PEG-OligoLys (magenta) was injected for observation 7 hours later. Colocalization of these two colors is observed as white or cyan (encircled by yellow dotted lines). (F) Blood circulation profiles of PEG without OligoLys, and one- and two-arm-PEG-OligoLys. n = 4. Data are shown as means SEM.
The two-arm-PEG-OligoLys fluorescence signal at the sinusoidal wall gradually decreased and became almost undetectable at 6 hours or later after injection (Fig. 3, B and D), whereas one-arm-PEG-OligoLys remained localized to the sinusoidal wall even at 9 hours after injection, with a minimal decrease in the fluorescence intensity of the sinusoidal wall during the observation period (Fig. 3, A and C). Closer observation revealed that two-arm-PEG-OligoLys was progressively accumulated to the space between the hepatocytes (encircled with dotted lines in Fig. 3B) at 3 hours or later after injection, whereas one-arm-PEG-OligoLys exhibited an almost undetectable accumulation to that space even at 9 hours after injection. On the basis of its anatomical position, the space may correspond to the bile canaliculi, which collect the bile from hepatocytes for clearance through the bile ducts. To clarify this point, a fluorescent bile tracer, 5-carboxyfluorescein (CF), was injected 5 min before two-arm-PEG-OligoLys injection. The position of two-arm-PEG-OligoLys accumulation at 7 hours after injection was colocalized with that of CF, as observed in the white or cyan pixels in Fig. 3E, which resulted from the merging of green (CF) and magenta pixels (two-arm-PEG-OligoLys). These observations indicate the gradual biliary clearance of two-arm-PEG-OligoLys.
The clearance profile of one- and two-arm-PEG-OligoLys was additionally evaluated by observing their persistence in the blood circulation. While these two groups showed comparable blood circulation profile within 1 hour after injection, obvious differences were observed at 1 hour or later after injection (Fig. 3F); the blood concentration of two-arm-PEG-OligoLys gradually decreased, while that of one-arm-PEG-OligoLys remained almost constant. The blood concentrations of one- and two-arm-PEG-OligoLys fit the two-compartment model with high R2 values, in which the polymers were administered into the central compartment and subsequently distributed into a tissue compartment (fig. S1 and table S1). These two formulations showed a comparable distribution phase half-life of around 15 min, with a comparable distribution rate constant (k12). This is consistent with the observation that both formulations similarly showed rapid binding to hepatic sinusoids. On the other hand, the elimination phase half-life of one-arm-PEG-OligoLys (13.3 hours) was much longer than that of two-arm-PEG-OligoLys (5.7 hours), which may reflect the different clearance behaviors of these two groups. The blood circulation profile of PEG without OligoLys conjugation fits the one-compartment model with high R2 values and presented a long half-life (19.8 hours). Without binding to vessel walls, this formulation may lack a distribution phase.
To obtain further mechanistic insights into the different behaviors between one- and two-arm-PEG-OligoLys, these two formulations were coinjected into mice for IVCLSM observation of their distribution in the hepatic sinusoids after labeling one-arm-PEG-OligoLys with Alexa647 (fig. S2, red) and two-arm-PEG-OligoLys with Alexa594 (green). Both formulations showed comparable levels of liver sinusoidal accumulation at 5 min to 1 hour after injection (Fig. 4 and movie S1). This observation suggests that the binding affinity of these formulations to the sinusoids is comparable. In sharp contrast, fluorescence from two-arm-PEG-OligoLys in the sinusoidal wall became weak, especially 6 hours or later after injection, presumably through biliary clearance, while a strong fluorescence signal from one-arm-PEG-OligoLys was consistently observed in the wall. Eventually, the sinusoidal walls in the images gradually became red (one-arm-PEG-OligoLys), with green (two-arm-PEG-OligoLys) appearing in the presumable location of the bile canaliculi 6 hours or later after injection. This observation is consistent with that after the single injection of each formulation, with two-arm-PEG-OligoLys still gradually cleared in the presence of one-arm-PEG-OligoLys. Thus, one-arm-PEG-OligoLys may preserve the liver functionality of biliary clearance but failed to be cleared under these conditions.
Alexa647-labeled one-arm-PEG-OligoLys (red) and Alexa594-labeled two-arm-PEG-OligoLys (green) were coinjected from the tail vein. (A) IVCLSM imaging of the liver. Presumable regions of bile canaliculi are encircled with white dotted lines. (B to D) Intensity profiles of Alexa594 and Alexa647 in the white arrows shown in (A). (B) 0.5 min, (C) 5 min, and (D) 6 hours after injection.
Toward safe usage of two-arm-PEG-OligoLys, it is important to estimate its clearance rate. For this purpose, blood clearance profile of two-arm-PEG-OligoLys was observed under its continuous intravenous infusion. In this experiment, bolus intravenous injection of two-arm-PEG-OligoLys was performed at a dose of 1250 g per mouse, which is the same as that used throughout this study. Subsequently, two-arm-PEG-OligoLys was infused at the rate reduced in a stepwise manner, to find the rate that allows the blood level of two-arm-PEG-OligoLys to be constant. Under such condition, the infusion rate of two-arm-PEG-OligoLys would be balanced with its clearance rate. The blood level of two-arm-PEG-OligoLys was constant under the infusion rate of 1200 g/hour per mouse and gradually decreased under the rate of 630 g/hour per mouse (fig. S3). This result suggests that the clearance rate of two-arm-PEG-OligoLys was approximately 1200 g/hour per mouse. This clearance may occur mainly through the biliary pathway, as two-arm-PEG-OligoLys with molecular weight over 80 kDa is unlikely to be cleared through the renal pathway. Two-arm-PEG-OligoLys accumulation to the bile canaliculi was observed in intravital observation of the liver 3 hours or later after the injection (Fig. 3B). It is also worthy to note that the biliary clearance rate of two-arm-PEG-OligoLys (1200 g/hour per mouse = 240 pmol/min per mouse) is comparable with that of cationic drugs (100 to 1000 pmol/min per mouse), as reported previously (38).
We then checked hemolysis and change in major biomarkers related to liver and kidney functions to estimate potential acute toxicity of injected polymers. Two-arm-PEG-OligoLys, as well as one-arm-PEG-OligoLys, showed no ex vivo hemolytic activity (fig. S4) and no detectable changes in plasma levels of a general tissue damage marker [lactate dehydrogenase (LDH)], liver damage markers [aspartate aminotransaminase (AST) and alanine aminotransferase (ALT)], and kidney function markers [blood urea nitrogen (BUN) and creatinine (Cre)] after in vivo administration (table S2). On the other hand, non-PEGylated OligoLys induced a substantial level of hemolysis activity ex vivo and LDH release in vivo.
Together, the above results demonstrate that the clearance behavior of the PEGylated OligoLys was successfully controlled by fine-tuning of PEG configuration. PEGylated OligoLys formulations used for the transient stealth coating of liver sinusoidal wall should simultaneously meet the following two requisites: (i) sufficient and selective stealth coating of the liver sinusoidal wall for retargeting nanomedicines and (ii) ensured clearance from the sinusoidal wall for avoiding chronic disturbance of physiological functions due to accumulation of PEG-OligoLys in the body. As shown in Figs. 2 and 3, both one- and two-arm-PEG-OligoLys attached to the sinusoidal walls selectively, meeting requisite (i). Worth noting is that two-arm-PEG-OligoLys was able to be cleared from the sinusoidal wall to the bile in several hours, while one-arm-PEG-OligoLys persisted on the wall even after 9 hours of the observation period. This result indicates that one-arm-PEG-OligoLys does not satisfy requisite (ii), which may induce safety concerns of chronic accumulation toxicity. Thus, we selected only two-arm-PEG-OligoLys for further examination devoted to evaluate redirecting efficacy of nanomedicines, demonstrating the enhanced gene expression of polyplex micelle (PM) and adeno-associated virus (AAV) in target tissues as described in the following sections.
To evaluate the feasibility of the sinusoidal PEG coating strategy, we first selected PM loading plasmid DNA (pDNA) as a model nanomedicine (39, 40). PM was prepared by mixing pDNA with one-arm-PEG-poly(l-lysine) (PLys) block copolymers with a PEG Mw of 12 kDa and a PLys polymerization degree of 44, installed with thiol moieties in 50% of the lysine residues for environment-responsive cross-linking between the cationic segments of the block copolymers. The PM was composed of a PEG shell and a core containing condensed pDNA. Disulfide cross-linking in the core stabilizes PM in extracellular environments and is selectively cleaved in intracellular reductive environments for pDNA release. According to our previous report, despite the stealth and stabilized PM formulation, a large fraction of the PM was cleared from the blood circulation within 1 hour after systemic injection, with only 23% of the dose remaining in the blood at 1 hour after injection (40). Such a moderate level of stealthiness provides us with a good platform for the application of the sinusoidal PEG coating strategy to prolong the persistence of PM in the blood circulation.
PM showed a cumulant diameter of 112 nm with a polydispersity index (PDI) of 0.15 and an almost neutral -potential of 1.5 mV, suggesting the successful formation of the core-shell structure, composed of a PEG shell and a core containing condensed pDNA. First, PM loading Cy5-labeled pDNA was intravenously injected into the mice without two-arm-PEG-OligoLys injection for IVCLSM observation of PM behavior in the liver. PM showed sinusoidal entrapment as early as 5 min after injection, despite the fact that PM was PEGylated (Fig. 5, A and C). When two-arm-PEG-OligoLys was preinjected into the mice 5 min before the PM injection, the sinusoidal entrapment of the PM was effectively prevented even at 1 hour after injection (Fig. 5, B and D). This process was more obviously visualized by labeling both of two-arm-PEG-OligoLys and PM, using Alexa594 for two-arm-PEG-OligoLys and Cy5-labeled pDNA for PM (fig. S5 and movie S2). Meanwhile, under continuous observation, PM preinjected with two-arm-PEG-OligoLys exhibited sinusoidal attachment to some extent at 3 hours after injection. This result is consistent with the gradual clearance of two-arm-PEG-OligoLys from the sinusoidal wall 3 hours after injection (Fig. 3, B and D).
Two-arm-PEG-OligoLys was intravenously injected to coat liver sinusoidal wall with PEG, followed by the intravenous injection of PM loading pDNA 5 min later. (A and B) IVCLSM imaging of PM loading Cy5-labeled pDNA (red) in the liver without PEG coating of sinusoid (A) or with the coating (B). Intensity profiles of Cy5 in the white arrows in (A) and (B) are shown in (C) and (D), respectively [(C) without coating and (D) with coating]. (E) Blood circulation profiles of PM with or without PEG coating of sinusoidal wall. n = 4. (F) PM loading Luc-expressing pDNA was injected to tumor-bearing mice with or without preinjection of two-arm-PEG-OligoLys. Luc expression in the tumor was measured 2 days after injection. n = 4. Data are shown as means SEM. Statistical analysis was performed using unpaired two-tailed Students t test.
The effect of two-arm-PEG-OligoLys preinjection on PM clearance was further evaluated by observing the blood circulation profile of PM. Without two-arm-PEG-OligoLys preinjection, PM showed two phases of decrease in its blood concentration, with a rapid drop within 1 hour after injection, followed by a gradual decrease (Fig. 5E). The marked decrease in the PM blood concentration could be attributed to its tissue distribution, including the sinusoidal entrapment, as shown in Fig. 5, A and C. Such rapid PM clearance from the blood was effectively prevented by two-arm-PEG-OligoLys preinjection, presumably via the prevention of sinusoidal PM clearance, as shown in Fig. 5, B and D.
These promising results motivated us to use our strategy for gene transfection at the tumor site, as the PM formulation used in this study provided successful outcomes in the antiangiogenic treatment of cancer in our previous reports (41, 42). PM loading luciferase (Luc) pDNA was intravenously injected into the mice bearing C26 murine colon carcinoma, 5 min after preinjection of two-arm-PEG-OligoLys. Two-arm-PEG-OligoLys preinjection resulted in a more than 10-fold increase in Luc expression efficiency in the tumor compared with the PM injection without two-arm-PEG-OligoLys preinjection (Fig. 5F). The enhanced transfection expression efficiency of PM in the tumor after two-arm-PEG-OligoLys preinjection could be attributed to the avoidance of PM sinusoidal entrapment, which may result in enhanced tumor accumulation of PM.
Last, we applied the two-arm-PEG-OligoLys preinjection approach to the administration of viral gene vectors, in which this technology is highly demanded. In particular, when organs other than the liver are targeted, sinusoidal entrapment of the vectors seriously hinders the ability of viruses to reach their target organs (14, 24), resulting in an increase in the viral dose, which then poses a safety problem. Although AAV is widely believed to be safe, high levels of toxicity have been observed in large animals after AAV administration at the dose that is required to obtain therapeutic levels of protein expression in the spine (43). Here, two-arm-PEG-OligoLys preinjection was performed 5 min before injection with AAV8 to prevent the sinusoidal clearance of AAV8 and to relocate it to the heart and skeletal muscles, which are promising target organs for the therapeutic application of AAV8 (44). Three weeks after the delivery of AAV8 expressing Luc, two-arm-PEG-OligoLys preinjection resulted in a decrease in the expression efficiency of Luc in the liver to 42% of the level observed without two-arm-PEG-OligoLys preinjection (Fig. 6A). This result suggests the successful prevention of AAV8 entrapment in the liver by the PEG coating of the sinusoidal wall using two-arm-PEG-OligoLys. Two-arm-PEG-OligoLys preinjection resulted in a significant increase in Luc expression in AAV8 target organs, a 4.3-fold increase in the heart (Fig. 6B), and a 2.3-fold increase in the skeletal muscles (Fig. 6C), respectively, presumably via the relocation of AAV8 from the liver sinusoids to these organs after sinusoidal PEG coating. This result demonstrates the effectiveness of our strategy in increasing the gene expression of viral vectors in their target organs, which will allow for a reduction in the dose of the vectors needed for gene therapy, thereby minimizing the safety concerns.
Five minutes after intravenous injection of two-arm-PEG-OligoLys for PEG coating of liver sinusoidal wall, AAV8 expressing Luc was intravenously injected. Three weeks later, Luc expression in the liver (A), heart (B), and skeletal muscle (C) was measured. n = 6. Data are shown as means SEM. Statistical analysis was performed using unpaired two-tailed Students t test.
An important feature of two-arm-PEG-OligoLys for future clinical applications is its transient binding profile to the liver sinusoidal walls with a gradual clearance to the bile, providing an advantage in terms of safety over one-arm-PEG-OligoLys, which persisted in the sinusoidal wall. To obtain mechanistic insight into the differences between one- and two-arm-PEG-OligoLys, first, the intrinsic biliary excretion profile of OligoLys without PEGylation was observed in the liver using IVCLSM. Non-PEGylated OligoLys exhibited a high accumulation to the presumable location of bile canaliculi, especially 3 hours or more after injection (fig. S6). This result indicates that OligoLys is intrinsically cleared to the bile, while this process is inhibited by single 80-kDa PEG chain conjugation to OligoLys but not by double 40-kDa PEG chain conjugation. Meanwhile, both one- and two-arm-PEG-OligoLys exhibited similar behavior in terms of their binding to the sinusoidal wall after coinjection (Fig. 4). Thus, binding affinity to the sinusoidal wall may not be a major factor for the differences between one- and two-arm-PEG-OligoLys. Two-arm-PEG-OligoLys was cleared to the bile even after coinjection with one-arm-PEG-OligoLys, indicating that one-arm-PEG-OligoLys preserves the liver functionality of biliary clearance. Even under such conditions, one-arm-PEG-OligoLys still failed to be cleared.
Although detailed molecular analyses should be performed in the future to fully explain such clearance behavior of one- and two-arm-PEG-OligoLys, it is worth proposing a possible mechanism, based on the following two hypotheses. (i) Sinusoidal walls are densely coated with PEG. (ii) Biliary clearance of PEGylated OligoLys occurs via the endocytotic pathway, especially clathrin-mediated endocytosis, which is dominant in LSECs (11). On the basis of the radius of gyration, the diameter of 40-kDa and 80-kDa PEG is around 20 and 30 nm, respectively, which is close to the typical size of clathrin-coated vesicle (50 to 200 nm) (45, 46). When cell membrane is densely coated with PEG, such large PEG chains would overlap with each other after curving of cell membrane in endocytosis, and such overlapping between PEG exclusion volume is entropically unfavorable based on a scaling theory (47, 48). Here, we estimated the effect of PEG configuration on the overlapping volume using mathematical modeling, by assuming one-arm-PEG-OligoLys as one sphere of 80-kDa PEG and two-arm-PEG-OligoLys as two spheres of 40-kDa PEG, which densely coat the plasma membrane with a hexagonal lattice structure, without overlapping. In this model, curving of cell membrane in 50- to 200 nm-sized vesicles induces overlapping of PEG chains, with 80-kDa PEG providing more than threefold larger volume of the overlap compared with 40-kDa PEG (note S1). This calculation suggests that long single PEG chain (80 kDa) may not represent a suitable cargo of endocytotic vesicles to facilitate biliary excretion, while separation of PEG chains into two segments is effective in avoiding this issue.
Such transient coating of liver sinusoidal walls with two-arm-PEG-OligoLys allowed us to relocate nonviral and viral gene vectors from the sinusoidal wall to their target tissues, thereby improving the gene transfection efficiency in the tissues. With the ability to improve nanomedicine pharmacokinetics, this approach can be used not only to enhance the effect of nanomedicines but also to reduce the dose required to obtain these effects, which is particularly important for reducing the toxicity of viral gene therapy. While clearance behavior of two-arm-PEG-OligoLys was evaluated in detail after its single bolus administration as well as under the continuous infusion for several hours (fig. S3), detailed examination of possible chronic toxicity due to polymer overloading upon multiple injections may be required in the future to translate this procedure of transient surface covering of sinusoids in clinics, because nanomedicines are administered repeatedly in many cases. Here, we faithfully focus on the configuration of PEG (linear versus two-arm branched) having the same total Mw of 80 kDa, yet optimization of total PEG Mw should also be addressed in the future for further optimal tuning of the liver sinusoidal coating to maximize the efficacy of nanomedicine therapy, with minimal influence on liver physiological functions. Our approach is versatile for combinational use with various nanomedicines, including synthetic and nature-derived nanomedicines, opening avenues for future nanotherapy and nanodiagnosis.
OligoLys with or without PEGylation was synthesized via the ring-opening polymerization (ROP) of N-trifluoroacetyl-l-lysine N-carboxyanhydride [l-Lys(TFA)-NCA, Chuo Kaseihin Co. Inc., Tokyo, Japan], as previously described for two-arm-PEG-OligoLys (49), one-arm-PEG-OligoLys (50), and non-PEGylated OligoLys (51). Briefly, for two-arm-PEG-OligoLys synthesis, two-arm--methoxy--amino-PEG [two-arm-PEG-NH2, Mn (number-average molecular weight) = 2 40 kDa, NOF Corporation, Tokyo, Japan] was used as a macroinitiator for the ROP of l-Lys(TFA)-NCA to obtain two-arm-PEG-OligoLys(TFA). The molecular weight distribution (Mw/Mn) of two-arm-PEG-OligoLys(TFA) was 1.04, according to size exclusion chromatography (SEC) (TOSOH HLC-8220; Tosoh Corp., Tokyo, Japan). The TFA groups were deprotected to obtain two-arm-PEG-OligoLys. The degree of polymerization (DP) of OligoLys in two-arm-PEG-OligoLys was 19, according to 1H nuclear magnetic resonance (NMR) spectrum (JEOL ECS 400; JEOL, Tokyo, Japan). For one-arm-PEG-OligoLys synthesis, one-arm-PEG-OligoLys(TFA) was synthesized using one-arm--methoxy--amino-PEG (one-arm-PEG-NH2, Mn = 83 kDa) as a macroinitiator of ROP of l-Lys(TFA)-NCA and exhibited Mw/Mn of 1.06 in SEC analysis. One-arm-PEG-OligoLys, obtained after the deprotection of TFA groups, showed an OligoLys DP of 21 in 1H NMR. For non-PEGylated OligoLys synthesis, OligoLys(TFA) was synthesized by ROP of l-Lys(TFA)-NCA using n-butylamine (TCI Chemicals Co. Ltd., Tokyo, Japan) as an initiator, followed by the deprotection of TFA groups to obtain OligoLys. The DP of OligoLys was 28, according to the 1H NMR spectrum. The fluorescence labeling of OligoLys with or without PEGylation was performed as previously described (49). Briefly, one- and two-arm-PEG-OligoLys and non-PEGylated OligoLys were labeled with a single molecule of Alexa dye at OligoLys at the main chain end the -NH2 group before deprotecting the TFA groups using the N-hydroxysuccinimide (NHS) ester of Alexa Fluor 594 or 647 (Thermo Fischer Scientific, Waltham, MA, USA), according to the manufacturers instructions. For injection, OligoLys with or without PEGylation, with or without fluorescence labeling, was dissolved in 10 mM Hepes buffer containing 150 mM NaCl (pH 7.3).
All animal experimental procedures were approved and conducted in compliance with the Institutional Guidelines for the Care and Use of Laboratory Animals as stated by the Animal Committee of the Innovation Center of NanoMedicine (iCONM).
All of the intravital observations in this study were performed using IVCLSM, an A1R confocal laser scanning microscope (Nikon Corp., Tokyo, Japan), connected to an upright ECLIPSE FN1 (Nikon Corp.), using the following settings. The pinhole diameter was set to obtain a 10-m optical slice. BALB/c mice (6 weeks old, female, 18 to 20 g, Charles River Laboratories Inc., Yokohama, Japan) were anesthetized with 2.5% isoflurane (Abbott Japan Co. Ltd., Tokyo, Japan) using a NARCOBIT-E Univenter 400 Anaesthesia Unit (Natsume Seisakucho Co. Ltd., Tokyo, Japan). The anesthetized mice were placed onto a temperature-controlled plate (Thermoplate; Tokai Hit Co. Ltd., Shizuoka, Japan) with the temperature set to 37C.
For the observation of blood vessels in the earlobe dermis, the earlobe was fixed using a drop of immersion oil beneath the coverslip. For the observation of the liver, the liver was surgically exposed and glued directly to the cover glass using a drop of oil. Fluorescence-labeled OligoLys with or without PEGylation was intravenously injected through a catheter inserted into the lateral tail vein slowly in approximately 30 s at the dose of 15 nmol per mouse (1.25 mg per mouse for one- and two-arm-PEG-OligoLys and 0.05 mg per mouse for non-PEGylated OligoLys). Throughout the study, the autofluorescence signal of liver parenchyma was excited using a 405-nm laser and detected using a 450/50-nm bandpass emission filter. Alexa594 was excited using a 561-nm laser and detected using a 595/50 bandpass emission filter. Alexa647 was excited using a 640-nm laser and detected using a 700/50-nm bandpass emission filter. A 40 objective lens was used for liver imaging, while a 20 objective lens was used for earlobe imaging. Images were processed using NIS-Elements software (Nikon Corp.) for the quantification of fluorescence intensity. The fluorescence intensity of each pixel in the line charts was calculated after subtracting the background fluorescence intensity, which was measured using the images obtained 10 s before sample injection.
CF diacetate (CFDA, TCI Chemicals Co. Ltd.) was intravenously injected at a dose of 0.2 mg/kg. Five minutes later, a liver image was obtained using IVCLSM, by exciting CFDA using a 488-nm laser and detecting the fluorescence using a 520/50-nm bandpass emission filter. Immediately after the CFDA imaging, two-arm-PEG-OligoLys was intravenously injected for liver imaging 7 hours later, as described in the previous section.
The blood circulation profile of fluorescence-labeled OligoLys with or without PEGylation was quantified by measuring the fluorescence intensity of the blood vessel lumen in the earlobe after injection of the samples, as described in our previous report (49). Briefly, the fluorescence intensity in the region of interest (ROI) in the vein was measured at each time point, followed by the subtraction of the background fluorescence intensity obtained 10 s before the injection. The value obtained for each time point was standardized with the maximum fluorescence intensity of the ROI during the observation period.
In the coinjection of one- and two-arm-PEG-OligoLys, a mixture of 1.25 mg per mouse of Alexa647-labeled one-arm-PEG-OligoLys and 1.25 mg per mouse of Alexa594-labeled two-arm-PEG-OligoLys was injected from the tail vein. The parenchymal autofluorescence and fluorescence signal from Alexa594 and Alexa647 was detected as described in the Intravital observation of earlobe and liver section. After subtracting the background fluorescence intensity, which was measured using the images obtained 10 s before the sample injection, the fluorescence intensity of Alexa594 and Alexa647 was standardized on the basis of the intensity of fluorescence in the blood vessel lumen at 30 s after injection, set to 100% in Fig. 4 (B to D). The attachment of one- and two-arm-PEG-OligoLys to the sinusoidal wall was almost unobservable at 30 s after injection (Fig. 4, A and B).
OligoLys with or without PEGylation was injected into the tail vein at the same dose as for intravital imaging above (1.25 mg per mouse for one- and two-arm-PEG-OligoLys and 0.05 mg per mouse for non-PEGylated OligoLys). Blood was collected from the mice 4 hours after injection to examine the plasma using a DRI-CHEM 7000i system (Fujifilm, Tokyo, Japan).
Mouse blood was centrifuged at 500g for 5 min to sediment the blood cells, followed by washing with phosphate-buffered saline (PBS; pH 7.4) twice. Red blood cells (RBCs) collected from 1 ml of the blood were suspended in 20 ml of PBS. One volume of OligoLys with or without PEGylation was added to 10 volumes of the RBC suspension. The final concentration of OligoLys with or without PEGylation was adjusted to 7.5 pM, which is the same as the calculated concentration of OligoLys in the blood when OligoLys injected at the dose used in intravital imaging above was evenly distributed in 2 ml of mouse blood. The mixture was incubated at 37C for 1 hour, followed by centrifugation at 500g for 5 min. The absorbance of the supernatant at 405 nm was measured using Microplate Reader Infinite M1000 Pro (Tecan Japan Co. Ltd., Kanagawa, Japan) to quantify the amount of hemoglobin. A mixture of one volume of Triton X-100 (20% v/v) and 10 volumes of RBC suspension was sonicated for use as a positive control (exhibits 100% activity of hemolysis). The absorbance value of each sample was compared to the value obtained for the positive control.
PEG-PLys, used for constructing PM as described in the following section, was synthesized via ROP of l-Lys(TFA)-NCA using PEG-NH2 (Mn = 12 kDa) (NOF Corporation) as a macroinitiator. The Mw/Mn of PEG-PLys(TFA) was 1.05 according to SEC. The DP of PLys in PEG-PLys was 44, based on the 1H NMR spectrum. The 1-imino-4-mercaptobutyl (IM) groups were introduced onto the side-chain -amino groups of the lysine units of the PLys segment in PEG-PLys [PEG-PLys(IM)] using 2-iminothiolane (Thermo Fischer Scientific), according to a previous report (39). The introduction ratio of IM in the total NH2 groups in the original PEG-PLys was 50%, according to the 1H NMR.
A pDNA expressing Luc, pCAG-Luc2, was constructed by cloning the Luc coding sequence of pGL4.13 vector (Promega, Madison, WI, USA) into the pCAG-GS vector (RIKEN BioResource Research Center, Tsukuba, Japan). PM was prepared from PEG-PLys(IM) and pCAG-Luc2 pDNA at [amino groups in PEG-PLys(IM) (N)] to [phosphate groups in pDNA (P)] (N/P) ratio of 2, as previously reported (39).
The dynamic light scattering (DLS) and -potential measurements were measured using a Zetasizer Nano ZS ZEN3500 (Malvern Instruments Ltd., Worcestershire, UK). For these measurements, the pDNA concentration was adjusted to 33.3 g/ml, dissolved in 10 mM Hepes buffer containing 150 mM NaCl for DLS measurement and in 10 mM Hepes buffer without NaCl addition for -potential measurements. The hydrodynamic diameter (DH) and PDI of PM were evaluated using DLS at a detection angle of 173 and a temperature of 25C using cumulant methods. The -potential was measured with electrophoretic light scattering at 37C using Smoluchowskis equation.
For injection, the pDNA concentration was adjusted to 100 g/ml with a final concentration of Hepes and NaCl of 10 and 150 mM, respectively.
For the intravital imaging of PM, pCAG-Luc2 pDNA was labeled with Cy5 using the Label IT Tracker Intracellular Nucleic Acid Localization Kit (Mirus Bio Corp., Madison, WI). PM loading Cy5-labeled pCAG-Luc2 pDNA was intravenously injected into the tail vein at the dose of 20 g per mouse 5 min after the intravenous preinjection of two-arm-PEG-OligoLys at a dose of 1.25 mg per mouse. The control mice were injected with 10 mM Hepes buffer containing 150 mM NaCl (pH 7.3) instead of two-arm-PEG-OligoLys solution before PM injection. Liver imaging and the evaluation of the blood circulation profile were performed, as described in the Intravital observation of earlobe and liver and Evaluation of blood circulation profile sections, respectively.
Murine colon adenocarcinoma 26 (C26) cells were obtained from the National Cancer Center (Tokyo, Japan) and cultured in high-glucose Dulbeccos modified Eagles medium containing 10% fetal bovine serum. C26 cells (5 106 cells per mouse) were inoculated into subcutaneous tissue in the right rear flank of BALB/c nu/nu mice (7 weeks old, female, Charles River Laboratories). Mice with tumors of approximately 100 mm3 were intravenously injected with PM loading 20 g of pCAG-Luc2 pDNA, with or without two-arm-PEG-OligoLys preinjection, as described in the previous section. Tumors were harvested 48 hours after PM injection. The extracted tumor was homogenized using Multibeads Shocker in passive lysis buffer (Promega, Madison, WI, USA), followed by a Luc assay using a Luciferase Assay System (Promega) and Lumat LB9507 (Berthold Technologies, Bad Wildbad, Germany). The luminescence intensity values were normalized to the total protein amount in the homogenates determined by the Micro BCA Protein Assay Reagent Kit (Thermo Fischer Scientific). The values were presented after subtracting the background values obtained from the tumors harvested from mice without PM injection.
BALB/c mice (6 weeks old, female, Charles River Laboratories) were intravenously injected with 1.25 mg of two-arm-PEG-OligoLys, followed by the injection of AAV8 encoding firefly Luc driven by the CMV-IVS promoter (Vector Biolabs, Malvern, PA, USA) at the dose of 2.5 1011 viral genomes per mouse, sequentially at 5-min intervals. For the control mice, 10 mM Hepes buffer containing 150 mM NaCl (pH7.3), instead of two-arm-PEG-OligoLys, was injected before the AAV injection. Three weeks after AAV8 injection, the liver, heart, and muscles from the backside were excised. The Luc assay and data were analyzed as described in the previous section for the quantification of Luc expression in the tumor tissue.
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
Acknowledgments: We thank M. Kuronuma and Y. Satoh (Kawasaki Institute of Industrial Promotion) for technical assistance. Funding: This research was supported financially by the Japan Science and Technology Agency (JST) through the Center of Innovation (COI) Program [Center of Open Innovation Network for Smart Health (COINS) (grant number JPMJCE1305)], Research on the Innovative Development and the Practical Application of New Drugs for Hepatitis B from the Japan Agency for Medical Research and Development (AMED) (JP17fk0310111 to K.K.), and Grants-in-Aid for Scientific Research (B) (18 K03529 to S.U.) and for Early-Career Scientist (18 K18393 to A.D.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). Author contributions: A.D., S.U., and K.K conceived the idea, designed all the experiments, and wrote the manuscript. A.D. performed all the experiments. K.T. helped with the IVCLSM experiments. S.A. performed the pharmacokinetic analysis. H.K. assisted with the virus experiment. S.F. helped with synthesis of the oligocations. J.L., S.O., T.A.T., X.L., K.H., and Y.M. contributed in the other experiments. K.O. discussed the experimental data. S.U. and K.K. supervised the whole project. Competing interests: K.K. is a founder and a scientific advisor of AccuRna Inc. The remaining authors declare that they have no conflict of interests. PCT patent pending: Kawasaki Institute of Industrial Promotion (K.K., S.O., S.U., K.H., A.D., and K.T). Date: 12 March 2019; serial numbers: PCT/JP2019/009919. JP patent pending: Kawasaki Institute of Industrial Promotion (K.K., S.O., S.U., K.H., and K.O). Date: 19 November 2019; serial numbers: JP2019/520319. Data and materials availability: All experimental data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested are from the authors.
MagForce : Publishes Financial Results for the Year 2019 and Operative Highlights – Marketscreener.com
DGAP-News: MagForce AG / Key word(s): Annual ResultsMagForce AG Publishes Financial Results for the Year 2019 and Operative Highlights (news with additional features)
30.06.2020 / 08:30 The issuer is solely responsible for the content of this announcement.
MagForce AG Publishes Financial Results for the Year 2019 and Operative Highlights
Europe: Successful implementation of the expansion strategy - significant increase in treatment numbers of brain tumor patients (post period)
USA: FDA approval to proceed with its streamlined trial protocol for the next stage of pivotal U.S. single-arm study for the focal ablation of intermediate risk prostate cancer with the NanoTherm therapy system received - first patients enrolled (post period)
Berlin, Germany, and Nevada, USA, June 30, 2020 - MagForce AG (Frankfurt, Scale, XETRA: MF6, ISIN: DE000A0HGQF5), a leading medical device company in the field of nanomedicine focused on oncology, published today its financial results as of and for the year ended December 31, 2019 as well as operative highlights.
In 2019, MagForce achieved important corporate goals and made decisive progress in its development.
Europe: Faster and easier access to treatment with NanoTherm therapy for brain tumor patients
All Company activities in Europe are aimed at further increasing awareness of the NanoTherm therapy system and making the therapy available to patients: MagForce successfully introduced its 'NanoTherm Therapy School' series, a practice-oriented, unique, multifaceted application training for the use of NanoTherm Therapy in treating brain tumors developed in close partnership with leading experts Prof. Dr. Walter Stummer, PD Dr. Dr. Oliver Grauer, University Hospital Mnster, and PD Dr. Johannes Wlfer, Hufeland Klinikum GmbH Mhlhausen. Targeted towards medical professionals working in the field of neuro-oncology, the training series aims at certifying surgeons in the use of the Company's innovative NanoTherm technology. Two sessions were successfully completed in 2019. In Lublin, Poland, the first treatment center outside Germany, and in Zwickau, another NanoTherm therapy center in Germany, the Parcelsus Clinic, were opened.
These achievements already led to enormously increased corporate sales in the first quarter of 2020. MagForce continues to successfully drive forward its European roll-out strategy in the current fiscal year.
USA: High medical need for a new less invasive, effective and well-tolerated treatment option for prostate cancer in the USA
In 2019, MagForce USA, Inc. successfully completed the first stage of its pivotal clinical US study for the focal ablation of intermediate risk prostate cancer. During this Stage 1 a standardized clinical procedure was validated and developed: MagForce USA had to develop a new procedure by which the NanoTherm is placed in a clinical targeted volume (CTV) of less than 2 to 4cc of volume in the human prostate and provides for a true focal ablation therapy. By modifying the thixotopic nature of the NanoTherm, an increase in viscosity of 100 times was achieved, which allowed NanoTherm to remain at the reverse biopsy instillation site and allow time for the NanoTherm conjugation to occur stabilizing the NanoTherm particles in the CTV. Initial findings showed only minimal treatment related side effects, which were tolerable and similar to those commonly associated with biopsies. The ablation analysis showed very well-defined ablation and cell death in the region of the nanoparticle deposit as it was observed with the previous pre-clinical studies.
The purpose of this focal ablation registration study, which will enroll up to 120 men in a single arm study, is to demonstrate that NanoTherm therapy can focally ablate cancer lesions with minimal side effects for patients who have progressed to intermediate risk prostate cancer stage and are under active surveillance. By destroying these cancer lesions, it is anticipated that patients will be able to remain in Active Surveillance Programs and avoid definitive therapies such as surgery or whole gland radiation with their well-known side effects as long as possible.
In April 2020 the FDA approved a streamlined trial protocol, for the next stage of the Company's pivotal U.S. study with the NanoTherm therapy system for the focal ablation of intermediate risk prostate cancer . The next stage of the clinical trial is being initiated with three well-respected urological centers in Texas, Washington and Florida who actively enrolled patients in Stage 1. In the meantime first patients were enrolled and MagForce is starting treatments.
Results of operations, net assets and financial position
In the financial year, revenues amounted to EUR 840 thousand (previous year: EUR 67 thousand). Revenues were generated from the commercial treatment of patients with NanoTherm therapy in Germany in the amount of EUR 47 thousand (previous year: EUR 66 thousand) and NanoTherm deliveries to subsidiaries in the amount of EUR 793 thousand (previous year: EUR 0 thousand).
Other operating income amounted to EUR 904 thousand. While the previous year was dominated by the extraordinary effect of the intra-group transfer of shares in MagForce USA Inc., which resulted in the realization of hidden reserves in the amount of EUR 13,895 thousand, there was no further transfer of shares in 2019. Consequently, other operating income decreased from EUR 14,909 thousand by EUR 14,005 thousand compared to previous year. Other operating income mainly consists of costs recharged to subsidiaries for management services and other administrative services in the amount of EUR 545 thousand (previous year: EUR 561 thousand), the reversal of provisions in the amount of EUR 173 thousand (previous year: EUR 293 thousand), exchange rate differences in the amount of EUR 75 thousand (previous year: EUR 71 thousand) and income relating to other periods in the amount of EUR 47 thousand (previous year: EUR 7 thousand).
Personnel expenses of EUR 3,987 thousand (previous year: EUR 3,921 thousand) also include bonus payments.
Other operating expenses of EUR 3,371 thousand are EUR 197 thousand higher than in the previous year (EUR 3,174 thousand). The increase in other operating expenses is mainly due to higher impairment losses on interest receivables from the affiliated company MT MedTech Engineering GmbH that is funded by MagForce AG as well as higher patent costs.
While the previous year due to extraordinary effects showed a positive operating result of EUR 6,828 thousand, 2019 closed with a negative operating result of EUR 6,203 thousand. The positive operating result of the previous year is due to the extraordinary effect of the intra-group transfer of shares in MagForce USA Inc. with the realization of hidden reserves in the amount of EUR 13,895 thousand. Normalized for this effect, the Company would have reported a higher negative operating result of EUR 7,067 thousand in the previous year. Interest income of EUR 215 thousand was largely at the same level as in the previous year (EUR 231 thousand), while interest expenses fell by EUR 140 thousand from EUR 1,823 thousand to EUR 1,683 thousand. The reason for the decrease in interest expenses is lower interest on share price linked liabilities. The write-down of the contributions to fund the operations of the subsidiary MT MedTech Engineering GmbH amounted to EUR 1,058 thousand (previous year: EUR 877 thousand). The partially contrary effects resulted overall in only a slight increase in the negative financial result of EUR 58 thousand from EUR 2,468 thousand to EUR 2,526 thousand.
The year 2019 closed with a net loss for the year of EUR 8,731 thousand. The net income of EUR 4,358 thousand in the previous year was due to the extraordinary effect of the intra-group transfer of shares in MagForce USA Inc. with the realization of hidden reserves amounting to EUR 13,895 thousand. Normalized for this effect the previous year ended with a net loss of EUR 9,537 thousand.
Cash flow from operating activities amounted to EUR -5,671 thousand (previous year: EUR - 7,106 thousand). The cash outflow from operating activities was derived indirectly from the net loss for the period. The cash outflows mainly relate to the financing of operating activities.
Cash flow from investing activities amounted to EUR - 1,941 thousand (previous year: EUR - 1,370 thousand) and mainly related to the contributions made in the reporting year to provide financial support for the subsidiary MT MedTech Engineering GmbH as well as the construction of mobile NanoActivator therapy centers and the expenses for the preparation of the technical documentation of the MagForce products.
Cash flow from financing activities amounted to EUR 6,286 thousand (previous year: EUR 9,304 thousand) and is mainly attributable to the proceeds from the capital increase and the stock options exercised. The payments were offset by cash outflows in the form of interest payments.
At the end of the year, the freely available liquidity amounted to EUR 167 thousand (previous year: EUR 1,494 thousand.
Outlook and financial prognosis of 2020
For the year 2020, the following focal points are planned for the Company's development:
In Europe: Increase in the number of commercially treated patients in Poland and Germany, initiation of further placements of NanoActivator devices in Germany and other European countries for the treatment of brain tumors, and the establishment of an efficient reimbursement procedure in Germany and the target countries for NanoTherm therapy in combination with surgery, radiation or chemotherapy as well as continuation and establishment of the "NanoTherm Therapy School" as application training for the use of NanoTherm therapy with the aim of certifying surgeons using the innovative NanoTherm technology.
In the USA: Conducting the second stage of the pivotal trial of NanoTherm therapy in the indication prostate cancer for the territory of the USA by the subsidiary MagForce USA, Inc., preparations for the commercialization of NanoTherm therapy for the treatment of prostate cancer in the USA. Further the completion of the development of an ambulatory NanoActivator device for the focal treatment of prostate cancer.
The impact of the outbreak of the corona pandemic on the future core activities described above cannot be predicted in detail at this time.
The Company expects a significant increase in the number of patients treated in both Germany and Poland in the financial year 2020, which will have a positive effect on earnings.
MagForce expects an increase in production volumes of NanoTherm to supply the US subsidiary due to the continuation of its pivotal trial and preparations for commercialization in the USA, as well as for the treatment of patients in Germany and Poland. The production of NanoActivator ambulatory devices will take place depending on the progress of the prostate study in the USA.
The expected revenues will not be able to compensate the expenses due to the continuation of the expansion strategy and the associated initiation of treatment series to obtain reimbursement as well as the necessary expansion of commercialization activities, so that a significant operating loss is also expected for the financial year 2020.
Management expects higher debt financing of the business activities and an associated increase in the negative financial result, provided that there are no significant opposing effects from share price linked debt components.
On June 24, 2020 MagForce signed an agreement with U.S. investment firm Yorkville Advisors Global LP for a growth financing via convertible notes of up to EUR 15 million to be drawn in probably up to five tranches. The first tranche of EUR 2.5 million each is expected to be drawn shortly. This financing enables MagForce to further implement its commercial strategy and support the roll-out of its NanoTherm Therapy treatment system.
It can be assumed that the corona pandemic will affect our forecasts, the exact scope of which cannot be estimated at this time.
About MagForce AG and MagForce USA, Inc.MagForce AG, listed in the Scale segment of the Frankfurt Stock Exchange (MF6, ISIN: DE000A0HGQF5), together with its subsidiary MagForce USA, Inc., is a leading medical device company in the field of nanomedicine focused on oncology. The Group's proprietary NanoTherm(R) therapy enables the targeted treatment of solid tumors through the intratumoral generation of heat via activation of superparamagnetic nanoparticles.
NanoTherm(R), NanoPlan(R), and NanoActivator(R) are components of the therapy and have received EU-wide regulatory approval as medical devices for the treatment of brain tumors. MagForce, NanoTherm, NanoPlan, and NanoActivator are trademarks of MagForce AG in selected countries.
For more information, please visit: http://www.magforce.com. Get to know our Technology: video (You Tube)Stay informed and subscribe to our mailing list.
This release may contain forward-looking statements and information which may be identified by formulations using terms such as "expects", "aims", "anticipates", "intends", "plans", "believes", "seeks", "estimates" or "will". Such forward-looking statements are based on our current expectations and certain assumptions, which may be subject to a variety of risks and uncertainties. The results actually achieved by MagForce AG may substantially differ from these forward-looking statements. MagForce AG assumes no obligation to update these forward-looking statements or to correct them in case of developments, which differ from those, anticipated.
Contact:Barbara von FrankenbergVice PresidentCommunications & Investor RelationsT +49-30-308380-77E-Mail: email@example.com
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Development of safe liver sinusoid coating agents to increase the efficacy of gene therapy – Science Codex
5pm on June 26, 2020 - Kawasaki/Japan: The Innovation Center of NanoMedicine (iCONM), the National Institute for Quantum Science and Technology (QST), and the University of Tokyo jointly announced that a reagent for the selective and safe coating of the liver sinusoidal walls to control the clearance of gene therapy drugs was successfully developed. The contents of this research will be published in Science Advances by the American Association for the Advancement of Science (AAAS) at 2:00 pm on June 26, east coast of the United States (Japan standard time: 3:00 am on 27th): A. Dirisala, S. Uchida, K. Toh, J. Li, S. Osawa, T. A. Tockary, X. Liu, S. Abbasi, K. Hayashi, Y. Mochida, S. Fukushima, H. Kinoh, K. Osada, Kazunori Kataoka, "Transient stealth coating of liver sinusoidal wall by anchoring two-armed PEG for retargeting nanomedicines".
Recently, gene therapies have been successively approved in Europe, US, and Japan, and are expected to provide novel therapeutic options for cancer, chronic diseases, acquired and inherited genetic disorders. Whilst this is promising, in reality, when gene therapy drugs are systemically administered to living organisms, they are rapidly eliminated and metabolized in the liver, thus impeding the delivery of a sufficient amount to the target organs and raising the toxicity concerns. This elimination by the liver is caused by the adsorption of the gene therapy drugs to the vascular wall of the liver sinusoid, which is an intrahepatic capillary. To overcome this issue, we conceived to selectively coat the liver sinusoidal wall using polyethylene glycol (PEG). However, a long-term coating may impair the normal physiological functions of the liver, and therefore the coating should be transient. In addition, coating needs to be selective for liver sinusoids, as coating the blood vessels throughout the body would not only cause adverse effects but also decrease the delivery amount of gene therapy drugs to target organs. Towards this end, we have developed a coating agent with two-armed PEG conjugated to positively charged oligolysine, which demonstrated the selective coating on the liver sinusoidal wall, the first-of-its-kind strategy in the world. Interestingly, the coating with two-armed PEG was excreted into bile within 6 hours after binding to sinusoidal walls, while the coating with single chain of linear PEG bound to oligolysine persisted in the walls for a long time. In this way, the precise molecular design was necessary to achieve a transient coating.
This coating was subsequently applied to boost the delivery efficacy of gene therapy drugs. Adeno-associated virus (AAV) is widely used for viral gene therapy drugs, and its serotype 8 (AAV8) targets myocardium and skeletal muscles. When AAV8 was administered after prior coating of two-armed PEG to the liver sinusoidal wall, the transfer of AAV8 to the liver was suppressed, and as a result, the gene transfer efficiency into the myocardium and skeletal muscles was improved by 2 to 4 times. This approach is promising for the treatment of muscular dystrophy. In addition, we expanded the use of our strategy to virus-free gene delivery systems, which allows more economically attractive and safe gene therapy. We have been working on non-viral gene therapy for malignant tumors using plasmid DNA-equipped smart nanomachine for over 10 years. When the coating agent was used for this system, the adsorption of nanomachines to the sinusoidal wall was suppressed, resulting in an approximately 10-fold improvement in DNA transfer efficiency to colon cancer. As described above, we have succeeded in boosting the activity of gene therapy drugs while ensuring safety by using the coating agent developed this time.
The above findings are summarized as follows:
- The coating agent with two-armed PEG selectively coated the liver sinusoid wall for several hours and was then excreted in the bile.
- The coating agent with single chain of linear PEG is not excreted in bile and coated the liver sinusoidal wall for more than 9 hours, which raises a safety concern.
- The coating agent with two-armed PEG had selectivity for the liver sinusoid wall, without coating the blood vessels in the connective tissues.
- The coating agent improved the gene transfer efficacy to the myocardium and skeletal muscles using the AAV vector by 2 to 4 times, and the gene transfer efficiency to colorectal cancer using DNA-loaded smart nanomachines by 10 times.
- As a result, our approach is expected to allow for improving the effect of gene therapy drugs and reducing their dose needed to obtain therapeutic outcome, which will lead to the reduction of medical cost and adverse event opportunities.