Tracing the Scientific Method SWOT Analysis of Innovation Chart

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  • Interpret and critically analyze primary scientific literature to assess the validity and reliability of scientific results and evaluate the conclusions drawn from these data
  • Demonstrate proficiency in scientific principles, techniques and applications in the life sciences to evaluate experimental design and determine compliance with standards of protocol and ethical practice
  • Effectively communicate scientific principles, concepts, methods, and research findings based on critical analysis of primary literature, industry reports, and other life sciences resources
  • Pose vital and relevant scientific questions to identify problems, challenges, and opportunities for the development of innovative products and services in the life sciences

I. Paper Format (10 points)

A. Paper label and title page (4):
1. Label your paper as follows: First and last name – SWOT (e.g., Sharon Brown - SWOT) (2)
2. Title page (name, instructor, title, due date) (2)
B. Sections of paper labeled properly (1)
C. Type double-spaced, 11-point, Times New Roman font paper with 1-inch margins (2)
D. 3-4 pages (not including title page and reference page) (3)

II. Paper Content (70 points) SWOT Analysis - Please follow the instructions, labeling each section of your paper according to the headings and subheadings for A and B below.A. Introduction of the innovation (20 points)
1. Trace the scientific method (5) – identify the observations, question, hypothesis, experiment, results, and conclusion of primary article B.
2. Eureka! moment (5) - In 4-5 sentences, describe a Eureka! moment - an aspect of the research
(introduction, materials, methods, results, discussion, conclusion) that stood out as you read primary article B which you believe could lead to an innovative technique, project, product, or service in the life sciences.
3. Innovation (5) – Based on the Eureka! moment you have identified, fully and specifically explain an innovative technique, service, product, or research. The innovation must be a technique, project, product, or service that you come up with on your own – not one presented in primary article B.
4. Contribution (5) - explain the ways in which your innovative method, research, product, or service can contribute to the biotechnology field.
B. SWOT Analysis of the innovation (50 points)
1. SWOT Analysis Chart (40)
a. Based on your proposed innovative method, research, product, or service, and the nature of your biotechnology company, fill in the SWOT analysis chart found below.
b. You must fill in at least five responses for each of the four categories.

2. SWOT Analysis follow-up (10)
a. How you can use the identified strengths to take advantage of your opportunities? (2.5)
b. How can the identified strengths combat the threats that are in the market? (2.5)
c. How can the identified external opportunities help you combat the identified internal weaknesses? (2.5)
d. How can you minimize the weaknesses so that you can avoid the threats that you identified? (2.5)

SWOT ANALYSIS CHART – FILL IN AND EMBED IN YOUR PAPER

Internal
1. Strengths - (10) List 5 internal resources of your company that could contribute to the success of your innovation

2. Weaknesses - (10) List 5 internal factors about your company that could hinder the success of your innovation

External
3. Opportunities (10) List 5 external factors that could contribute to the success of your innovation

4. Threats (10) - List 5 external threats that could hinder the success of your innovation

III. Paper References (15 points)
A. Use at least 10 references (5) DO NOT USE WIKIPEDIA
B. In-text references in APA format (5) (All references should be cited throughout the paper)
C. End references in APA-format (5)
D. If no references are cited, a grade of zero for this paper will be assigned.


IV. Paper Grammar (5 points) Spelling, punctuation, capitalization, sentence construction, and paragraph construction will be considered in the grading of this assignment.


V. Presentation Submission
A. Please label your paper with your first and last name followed by SWOT - for example, Sharon Brown – SWOT. B. Submit the assignment as a Word Document in the appropriate assignment folder. Do not submit this assignment as a PDF file – it will not be graded.

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SWOT ANALYSIS APPLIED TO THE LIFE SCIENCES BIOLOGY 495 SHARON BROWN, PH.D. WHAT IS A SWOT ANALYSIS? • SWOT stands for Strengths, Weaknesses, Opportunities, Threats. A SWOT analysis is a simple but useful framework for analyzing your organization's strengths and weaknesses, and the opportunities and threats that you face. • A SWOT analysis is one of the most commonly used strategic planning techniques. • The primary use of a SWOT analysis is to provide structure to, or summarize, your innovative analysis. This technique can also be used in decision making, to help determine which of several options is better. WHY USE A SWOT ANALYSIS IN THE LIFE SCIENCES? • A SWOT analysis will help summarize your understanding of the major issues identified in your innovative analysis • Your innovative analysis will focus on identifying the strengths, weaknesses, opportunities and threats in three key strategic environments. Three strategic environments: • Your internal environment • Your industry environment • Your macroenvironment STRENGTHS Strength refers to a core competency of your business where your business has a competitive advantage TANGIBLE INTERNAL STRENGTHS: These tend to be strengths that can be precisely identified, measured or realized •Physical assets, including plant or facilities and equipment •Assets you have in your team, such as knowledge, education, network, skills, and reputation •Long-term rental or business contracts •Unique or market-leading products •Financial resources to fund change or change management of your business •Competitive cost advantages •Advanced technologies or information systems •High volume business production •Scalability of business or products INTANGIBLE NTERNAL STRENGTHS: These tend to be strengths that cannot be physically touched or physically measured •Brand or product recognition •Location •Business reputation—Are you a market leader in your industry? •Goodwill and customer relationship management •Business/Supplier relationships •Strong employee relationships •Strategic business alliances or partnerships •Intellectual property rights or patents •Advertising strategy or process •Level of experience in your field •Level of management experience in your workforce •Industry knowledge superiority •Industry associations that give competitive advantage •Innovative practices within your business WEAKNESSES A weakness refers to a core competency of your business where your competitor has a competitive advantage when it comes to customer value propositions TANGIBLE INTERNAL WEAKNESSES: These tend to be weaknesses that can be precisely identified, measured or realized • Old or outdated plant and equipment • Lack of qualified personnel • Narrow product line • Insufficient financial resources to fund necessary changes • High operating costs • Inferior technology • Low volume and restricted scalability TANGIBLE EXTERNAL WEAKNESSES: These tend to be weaknesses that cannot be physically touched or physically measured • • • • • • • • • • Weak or unrecognizable brand Weak or unrecognizable image Poor relationships with your customers Poor relationships with your suppliers Poor relationships with your employees Marketing failing to meet objectives Manager inexperience Ineffective research & development Insufficient industry knowledge Meager innovative skills OPPORTUNITIES An opportunity is an environmental condition in your macro or industry environments that can improve your organization's competitive position relative to that of your competitors. When completing your analysis, you will find that your opportunities generally fall under two categories. INDUSTRY OPPORTUNITIES: These are opportunities in your industry environment and generally reduce the level of price competition in your industry. •Expansion of your product range •Diversification of your business interests •Growth in your customer's field •Growth in your supplier's field •Expansion of your customer base •Placid competition •Export opportunities •Products or service growth MACRO OPPORTUNITIES: These opportunities are in the broader environment that generally impacts all businesses in your region. •Favorable changes to legislation •Favorable changes to any import/export constraints •Favorable economic outlook •Favorable cultural shifts •Technology that your business can embrace and utilize, such as Ecommerce or Internet sales THREATS A threat is a forecasted environmental condition that is out of your control and has the potential to harm your business‘s profitability. INDUSTRY THREATS: These threats are related to an increase in the competition in your industry or a reduction in market size reduce your business‘s profitability. • • • • • Low cost imports: The threat of low-cost imports affects almost any manufacturer in the developed world Consumer ability to shift to a substitute product and changing demand for substitute products Slow market growth or decline in market size Shifts in customer or supplier buying power: The changing needs of buyers (customers) MACRO THREATS: These threats affect all industries in your region and result in risk of reduced profitability. • • • Shifts in foreign exchange rates that impact imports or exports: Demographic changes: The aging workforce makes it difficult to hire skilled workers in many developed countries. Industry Regulations: increasing regulations and increased costs needed to administer these new regulations. SWOT SUMMARY CONCLUSION: WHAT’S NEXT? • • • • With your SWOT analysis complete, you’re ready to convert it into real strategy. After all, the exercise is about producing a strategy that you can work on during the next few months to begin work your innovation. You will need to look at your strengths and figure out how you can use those strengths to take advantage of your opportunities. Then, look at how your strengths can combat the threats that are in the biotechnology market. Use this analysis to produce a list of actions that you can take. You will also want to analyze how external opportunities might help you combat your own, internal weaknesses. Can you also minimize those weaknesses so you can avoid the threats that you identified? The next step is to conduct a futuring analysis to see how your proposed innovation fits into the future of biotechnology. www.nature.com/scientificreports OPEN Received: 16 July 2018 Accepted: 16 January 2019 Published: xx xx xxxx Polymeric Engineering of Nanoparticles for Highly Efficient Multifunctional Drug Delivery Systems Beatrice Fortuni1, Tomoko Inose2, Monica Ricci1, Yasuhiko Fujita3, Indra Van Zundert1, Akito Masuhara4, Eduard Fron1, Hideaki Mizuno1, Loredana Latterini5, Susana Rocha1 & Hiroshi Uji-i1,2 Most targeting strategies of anticancer drug delivery systems (DDSs) rely on the surface functionalization of nanocarriers with specific ligands, which trigger the internalization in cancer cells via receptor-mediated endocytosis. The endocytosis implies the entrapment of DDSs in acidic vesicles (endosomes and lysosomes) and their eventual ejection by exocytosis. This process, intrinsic to eukaryotic cells, is one of the main drawbacks of DDSs because it reduces the drug bioavailability in the intracellular environment. The escape of DDSs from the acidic vesicles is, therefore, crucial to enhance the therapeutic performance at low drug dose. To this end, we developed a multifunctionalized DDS that combines high specificity towards cancer cells with endosomal escape capabilities. Doxorubicinloaded mesoporous silica nanoparticles were functionalized with polyethylenimine, a polymer commonly used to induce endosomal rupture, and hyaluronic acid, which binds to CD44 receptors, overexpressed in cancer cells. We show irrefutable proof that the developed DDS can escape the endosomal pathway upon polymeric functionalization. Interestingly, the combination of the two polymers resulted in higher endosomal escape efficiency than the polyethylenimine coating alone. Hyaluronic acid additionally provides the system with cancer targeting capability and enzymatically controlled drug release. Thanks to this multifunctionality, the engineered DDS had cytotoxicity comparable to the pure drug whilst displaying high specificity towards cancer cells. The polymeric engineering here developed enhances the performance of DDS at low drug dose, holding great potential for anticancer therapeutic applications. Over the last few decades, the engineering of nanoparticles has given rise to significant breakthroughs towards the employment of nanomaterials in biomedical applications, such as cancer therapy, (bio-) chemical sensing, and bio-imaging1–5. In particular, mesoporous silica nanoparticles (MSNPs) have been widely applied as promising anticancer drug nanocarriers thanks to their biocompatibility, high loading capacity, chemical stability and straightforward synthesis/surface functionalization6–8. Unlike some other nanocarriers, MSNPs have not been translocated into the clinical stage yet9. However, the reasonable biocompatibility accomplished in vivo is extremely promising for a proximate Food and Drug Administration (FDA-) approval10. To promote the specific internalization of nanoparticles to certain cancer cells (cancer targeting), many strategies have been developed so far. These methods are mainly based on the employment of specific ligands, which can bind to receptors overexpressed in tumor cells and trigger particle internalization via endocytosis11–14. In this context, hyaluronic acid (HA) has gained increasing attention as targeting ligand due to its high affinity with CD44, a glycoprotein receptor overexpressed in many solid tumor cells (e.g. lung, breast, pancreatic, renal tumor), 1 KU Leuven, department of Chemistry, Celestijnenlaan 200G-F, Heverlee, 3001, Belgium. 2RIES Hokkaido University, Research Institute for Electronic Science, N20W10, Kita-Ward Sapporo, 0010020, Japan. 3Toray Research Center, Inc., 3-3-7, Sonoyama, Otsu, Shiga, 520-8567, Japan. 4Yamagata University, department of Engineering, Yonezawa, Yamagata, 992-8510, Japan. 5University of Perugia, department of Chemistry, Biology and Biotechnology, via Elce di sotto 8, Perugia, Italy. Correspondence and requests for materials should be addressed to B.F. (email: beatrice. fortuni@kuleuven.be) or S.R. (email: susana.rocha@kuleuven.be) or H.U. (email: hiroshi.ujii@kuleuven.be) Scientific Reports | (2019) 9:2666 | https://doi.org/10.1038/s41598-019-39107-3 1 www.nature.com/scientificreports/ www.nature.com/scientificreports in metastasis, as well as in cancer stem cells15. As being one of the main constituents of the extracellular matrix, HA exhibits high biocompatibility, which has enabled its FDA-approval for medical and cosmetic use16–18. The harmlessness of HA, allied with its effective targeting capability, encouraged its employment to selectively internalize HA-functionalized materials (HA-materials) in CD44-overexpressing cancer cells via receptor-mediated endocytosis19–26. In spite of the well-achieved cell-specific internalization, the control of the particle fate after overpassing the plasma membrane remains challenging, and existing strategies are still limited. In eukaryotic cells, external materials (such as nutrients, protein and lipids, as well as nanoparticles), taken up via endocytosis, are normally sorted out in endocytic vesicles (endosomes and lysosomes) and can eventually be ejected to the extracellular matrix via exocytosis27. Previous reports have shown that non-coated MSNPs co-localize with the endo-/lysosomes in the early stage of incubation28–31, and are quickly exocytosed, following this pathway32. Similarly, HA-coated MSNPs are internalized via CD44-mediated endocytosis and are subjected to same endocytic system, ending up in the acidic cellular compartments within few hours of incubation23,33, and being ejected via exocytosis within 48 h34. The endo-/exocytosis process represents one of the main hindrances of the DDSs in light of the limited cargo release in the intracellular environment. The low lysosomal pH (4.5–5.5 for normal cells and 3.5–5 for cancer cells) and the strong enzymatic activity might lead to drug degradation, possibly inhibiting its pharmaceutical activity35. The therapeutic efficiency can be further decreased by the fast exocytosis of the nanocarriers36. As the drug release normally occurs by slow diffusion, the DDS can be exocytosed to the extracellular matrix before releasing all its cargo, contributing to the low therapeutic performance (forcing the use of higher drug dose), as well as to chemotherapy side effects. Despite the major consequences in terms of therapy efficiency, the intracellular route of nanocarriers is often neglected in the development of novel DDS, and strategies to enable the escape from this endocytic route are very limited. To this end, the employment of cationic polymers, in particular polyethylenimine (PEI), is a promising strategy, as it is non-immunogenic and easier to scale up, compared to other agents, such as viral proteins and synthetic fusogenic peptides37,38. PEI is already widely used in DNA transfection for promoting the release of genetic material from the acidic vesicles and, thus, facilitating the incoming to the nucleus39,40. The use of DNA-PEI polyplexes, instead of pure DNA, was demonstrated to improve the gene expression efficiency up to 100-fold40,41. This enhanced gene expression can be associated to the so-called “proton sponge effect” of PEI42. Most specifically, thanks to the protonation of tertiary amines, PEI exhibits high buffering capability at low pH, promoting an influx of protons inside the acidic cellular compartments via ATPase proton pumps and the consequent rupture of the organelle membrane due to an osmotic imbalance. The proton sponge effect of PEI is a generally accepted hypothesis in literature, however, it is important to mention that this concept is still heavily debated43. Since the action mechanism of most anticancer drugs, e.g. doxorubicin (Dox), is based on its intercalation into DNA and complex formation with DNA-associated enzymes44, the same approach can be used to enhance the nuclear delivery of anticancer drugs. The main hindrance for the application of PEI on DDSs is its cytotoxicity, which can be, howbeit, drastically reduced by using a low molecular weight (0.5–5 kDa)45,46. So far, PEI has been used to functionalize MSNPs for the successful delivery of either siRNA/DNA or siRNA/doxorubicin to HEPA-1 and KB-V1 cells, respectively45,47. In these studies, the endosomolytic activity of the PEI layer was assumed but not verified. On the other hand, Yanes et al. demonstrated that the addition of a PEI layer can slow down the exocytosis rate of MSNPs, although no investigation on the intracellular distribution of the nanoparticles was performed48. To the best of our knowledge, a study on the intracellular sorting of PEI-coated nanocarriers, which provides an evidence of their endosomal escape, has never been reported. In this manuscript, we propose a facile method to functionalize mesoporous silica nanoparticles with a polymeric bilayer, which simultaneously combines the active targeting action of HA and PEI-mediated endosomal escape (Fig. 1). For therapeutic applications, any anticancer drug can be loaded in the particles. Here, we use Dox-loaded MSNPs and show that the combination of active targeting, endosomal escape and controlled drug release results in high therapy efficiency. The method presented paves the way for the development of the next generation highly efficient DDSs. Results and Discussion Preparation and characterization of multifunctional MSNPs. Due to their popularity as highly stable, low-cost and reasonably biocompatible nanocarriers, mesoporous silica nanoparticles (MSNPs) were chosen as model of nanoparticle for the application of the polymeric coating here proposed10,49. MSNPs were synthetized using the biphase stratification method developed by Shen et al., that yields particles with a pore size of ~2.8 nm50. Transmission electron microscopy (TEM) images of uncoated MSNPs clearly show a uniform mesoporous frame (Fig. 2a). The particles exhibit size and shape homogeneity, with no observable aggregates. As depicted in Fig. 2b, the mean diameter was estimated to be 120 nm. After the synthesis, MSNPs were loaded with rhodamine B (RhodB) or fluorescein isothiocyanate (FITC) for monitoring cellular uptake/trafficking, and with Dox for testing the drug release and the therapeutic effect in cancer mammalian cells. The successful loading of dye/drug inside the pores was verified by fluorescence microscopy (Supplementary Fig. S1a–c). In order to provide the DDS with endosomal escape capability, MSNPs were coated with PEI (~1.3 kDa). Besides their biocompatibility and high loading capability, MSNPs offer a negatively charged surface which facilitates any kind of electrostatic interaction-based functionalization. At physiological pH, primary and secondary amines of PEI are protonated (pKa 8–10, depending on the molecular weight of the polymer)51,52, whereas ~50% of hydroxyl groups on a silica surface are deprotonated (pKa ≈ 6.8)53. This ionization percentage enables the formation of a PEI shell on the MSNP surface via electrostatic interaction. The presence of the PEI layer on MSNPs was demonstrated by the drastic change in the zeta potential of the particles after the coating (from −38.2 mV to +37.7 mV, Fig. 2c). Scientific Reports | (2019) 9:2666 | https://doi.org/10.1038/s41598-019-39107-3 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Multifunctional drug delivery system based on MSNPs: particle synthesis and cellular trafficking. (a–c) Preparation of HAPEI-MSNP_Dox: (a) encapsulation of doxorubicin (Dox) inside mesoporous silica nanoparticles (MSNP_Dox); (b) coating with polyethylenimine (PEI) layer (PEI-MSNP_Dox); (c) surface grafting with hyaluronic acid (HA) (HAPEI-MSNP_Dox). (d–f) Cellular uptake and intracellular trafficking: (d) particle interaction with the plasma membrane via CD44-HA site-specific binding; (e) HAPEI-MSNP_Dox uptake via receptor-mediated endocytosis and wrapping in endosomes; (f) rupture of the endosomal membrane upon proton sponge effect of PEI and drug release into the cytoplasm. A schematic illustration representing functions and chemical interactions of each component is reported at left-bottom of the figure. Figure 2. Characterization of MSNPs and their surface modifications. (a) TEM image of bare MSNPs. (b) Size distribution of the MSNPs (Gauss distribution in red fitting). (c) Zeta potential measurements of MSNPs, PEIMSNPs and HAPEI-MSNPs. (d–f) FE-SEM images of MSNPs, PEI-MSNPs and HAPEI-MSNPs, respectively. Scientific Reports | (2019) 9:2666 | https://doi.org/10.1038/s41598-019-39107-3 3 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 3. Influence of surface modification on the cellular uptake of MSNPs. (a–f) Fluorescence images of A549 and NIH3T3 cells after incubation with MSNPs_RhodB, PEI-MSNPs_RhodB and HAPEI-MSNPs_ RhodB for 3 h. RhodB-loaded particles are shown in orange; DiO-stained plasma membrane is colored in green. The central panel displays an xy-plane within the cells, while the right and bottom panels show the yz and xz projections, respectively. (g) Mean intensity of the RhodB signal per μm3 of cell (n = 4 for each condition), error bars indicate ± SD, with ns = (p > 0.05), *(p < 0.05), **(p < 0.01) and ***(p < 0.001). Thanks to the abundance of amino groups, the presence of PEI on the surface of MSNPs allowed for a straightforward binding of the targeting agent, HA, without any extra chemical modification. The carboxylic group of HA was covalently linked to the amino group of PEI via carbodiimide crosslinking reaction23. The decrease of the electrokinetic potential from +37.7 to +4.2 mV after HA grafting onto the PEI coating indicates the successful functionalization of the particles with HA (Fig. 2c). Considering such a change of the electrokinetic potential upon HA grafting, an effect on the charge-based PEI coating cannot be excluded. On the other hand, no attachment of HA would occur without the presence of a PEI layer on the surface, suggesting that the electrostatic interactions between PEI and the silanol groups endure the HA grafting process. The presence of the polymeric layers was confirmed using high resolution field-emission scanning electron microscopy (FE-SEM). Representative images of bare MSNPS, MSNPS coated with PEI (PEI-MSNPs) and MSNPs functionalized with a bilayer of PEI and HA (HAPEI-MSNPs) are shown in Fig. 2d–f, respectively. While the PEI layer is barely visible in the FE-SEM images of PEI-MSNPs (Fig. 2e), after conjugation with HA, the edge contrast increases, enabling an easier visualization of the polymeric layers in Fig. 2f. It is important to note that during image acquisition the coating collapsed and detached from the silica surface as a consequence of exposure to high accelerating voltages (30 kV). Therefore, the thickness of the layers visible in Fig. 2e,f does not correspond to the exact thickness of the shells. The halo displayed in Fig. 2e,f was never observed for bare MSNPs (additional FE-SEM images of bare MSNPs and HAPEI-MSNPs in Supplementary Fig. S2). Cellular uptake: HA-mediated active targeting. In order to evaluate the targeting efficiency and the cell specificity of the external functionalization with HA, we monitored the cellular uptake of the different particles into two mammalian cell lines. Most specifically, RhodB-loaded MSNPs with different coatings were added to A549 cells (CD44-overexpressing cells, derived from human lung carcinoma)54 and NIH3T3 (mouse embryonic fibroblasts, lowly expressing CD44 receptors, defined as CD44-negative or CD44-inactive cells)55. Figure 3 shows typical fluorescence images of both cell lines after 3 h of incubation with the different nanoparticles, with no coating (MSNPs), only a PEI layer (PEI-MSNPs) or functionalized with both HA and PEI (HAPEI-MSNPs). In order to quantify the cellular uptake, the plasma membrane was stained with a Scientific Reports | (2019) 9:2666 | https://doi.org/10.1038/s41598-019-39107-3 4 www.nature.com/scientificreports www.nature.com/scientificreports/ membrane-incorporating fluorescent dye (DiO, shown in green in Fig. 3). While there was a minimal amount of bare MSNPs detected inside the cells (Fig. 3a,d,g), PEI-MSNPs show a 2-fold increase in cellular uptake, independently of the cell line (Fig. 3b,e,g). This is in agreement with previously published results45 and is linked to the positive charge of PEI, which boosts electrostatic interactions with the electronegative plasma membrane and facilitates particle internalization. The addition of HA minimizes the surface charge of the PEI coated nanoparticles and reduces this effect. Consequently, in NIH3T3 cells, the uptake of HAPEI-MSNPs is similar to that of bare MSNPs (Fig. 3f). Remarkably, incubation of A549 cells with HAPEI-MSNPs results in a 10-fold increase on the amount particles detected inside the cell (compared with bare MSNPs, Fig. 3c and g). The drastic discrepancy in HAPEI-MSNP uptake rate between NIH3T3 (Fig. 3f) and A549 cell lines (Fig. 3c) proves that the HA functionalization provides our DDS (HAPEI-MSNP) with high specificity towards CD44-overexpressing cancer cells. Intracellular trafficking: PEI-induced endosomal rupture. Previous reports have shown that bare and HA-functionalized MSNPs traffic through the endocytic pathway, ending up into lysosomes and, eventually, being exocytosed23,28,30,31,33. In order to evaluate the effect of both PEI coating alone and its combination with HA on the endosomal trafficking, A549 cells were incubated with FITC-loaded particles for 3, 24 and 48 h. It is important to mention that after 3 h of incubation, the medium was refreshed to discard the excess of particles, preventing further internalization. After the incubation period, lysosomes were stained using LysoTracker Red , a fluorophore linked to a weak base that is only partially protonated at neutral pH and is fluorescent only in acidic environments. Cells were imaged by fluorescence microscopy and the co-localization between the fluorescence signal of FITC-loaded nanoparticles and LysoTracker Red was determined using the Pearson’s correlation coefficient (PCC)56 analysis (PCC threshold values of the current study are reported in SI, Supplementary Fig. S3). Figure 4 displays representative images of A549 cells incubated with MSNPs with different coatings, after 3, 24 and 48 h. The particles are shown in green while the acidic compartments are presented in red. As a consequence, MSNPs trapped in endo- or lysosomes are displayed in yellow. After 3 h, MSNPs without any additional surface functionalization co-localized with the endo-/lysosomes (Fig. 4a). Even after 48 h, all the particles detected inside A549 cells were co-localized with acidic compartments, indicating that none of the bare MSNPs taken up by the cell was able to escape the endocytic pathway (Fig. 4c). Accordingly, the calculated PCC is constant over time (black line in Fig. 4j). Note that, since the internalization rate of MSNPs is relatively low comparted to that of HAPEI- and PEI-MSNPs, in order to get an appropriate comparison study of the intracellular distribution, A549 cells with relatively higher MSNPs uptake were selected to perform confocal imaging and subsequent PCC analysis. Within a time span of 3 h, the PEI coating does not induce a clear effect on the intracellular fate of the nanoparticles, with bare MSNPs and PEI-MSNPs displaying similar intracellular distributions and co-localization coefficients (Fig. 4a,d,j). In stark contrast, after 24 h, PEI-MSNPs are roughly equally distributed between cytoplasm and acidic cellular compartments (Fig. 4e). The associated mean PCC value drastically decreases from 0.64 (3 h) to 0.36 (24 h), implying a reduced linear correlation between the fluorescence signal of PEI-MSNPs and endo-/lysosomes. At this time point, a high heterogeneity in the intracellular localization was observed between different cells, explaining the large standard deviation (SD) of the mean PCC value. As depicted in Fig. 4f, after 48 h the majority of PEI-MSNPs are excluded from the acidic compartments, with PCC value dropping to 0.25. The ability of PEI-coated particles to escape from the acidic vesicles is attributed to the proton sponge effect of this polymer, which results in the rupture of the membrane organelles42. It is important to mention that the possible proton sponge effect of PEI does not change the pH of the endo-/lysosomes57, and has no effect on the staining of these organelles with LysoTracker probes. Consequently, a lower co-localization with the LysoTracker Red can be directly linked to endo-/lysosomal damage and/or rupture. A similar trend was observed with the multifunctionalized HAPEI-MSNPs. After 48 h the majority of the particles with a HA-PEI bilayer were not co-localized with acidic cellular compartments (Fig. 4i). However, the initial uptake and endosomal escape rate is very different. At 3 h of incubation, a considerable fraction of HAPEI-MSNPs had already escaped the acidic vesicles (Fig. 4g, mean PCC = 0.45), indicating an effect of the polymeric bilayer in the endosomal escape rate (PCC similar to that of PEI-MSNPs after 24 h, Fig. 4j). The fraction of particles co-localizing the acidic compartments markedly decreased after 24 h, when most HAPEI-MSNPs were found to be no longer entrapped inside the endo-/lysosomal vesicles (Fig. 4h). After 48 h, practically all HAPEI-MSNPs were distributed in the cytosol (Fig. 4i, mean PCC = 0.10), indicating a highly effective escape of HAPEI-MSNPs from the acidic compartments. The results obtained with fluorescence imaging were further validated by electron microscopy. For the TEM measurements, cells were incubated with differently functionalized particles for 3 h and fixed after 24 h (more info in SI, Supplementary Figs S4 and 5). In agreement with the fluorescence images acquired at this time point, TEM images show that bare MSNPs were clearly trapped inside the lysosomes, MSNPs coated with PEI alone were found to be distributed either in the cytoplasm or inside the endo/lysosomes, and MSNPs with a polymeric bilayer (PEI and HA) were detected mainly outside of the lysosomes (Supplementary Fig. S5). These results constitute the first irrefutable evidence that coating of nanoparticles with specific polymers induces the rupture of the endo-/lysosomes and further escape to the cytosol. Both fluorescence and electron microscopy images demonstrate an evident enhancement/acceleration of the endosomal escape efficiency with HA-PEI coating compared to using PEI alone. Further research is necessary to assess the mechanism behind this effect, although we speculate that it might be associated either to a faster uptake rate of the HAPEI-coated particles, thanks to the HA targeting, or, more generally, to the presence of an additional polymer. At low pH, the inclusion of an extra polymeric layer can, indeed, increase both the buffering capacity and the polymeric swelling, contributing to the destabilization of the endo-/lysosomal membrane43. ® ® ® Scientific Reports | (2019) 9:2666 | https://doi.org/10.1038/s41598-019-39107-3 5 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 4. Influence of surface modification on the intracellular trafficking of MSNPs at different time points. (a–i) Fluorescence images of A549 cells incubated with MSNPs_FITC (a–c), PEI-MSNPs_FITC (d–f) and HAPEI-MSNPs_FITC (g–i) after 3, 24 and 48 h of incubation. The lysosomes were stained using LysoTracker Red . Green channel (FITC-loaded particles), red channel (LysoTracker Red-stained endo-/lysosomes) and DIC merged images are shown. (j) Co-localization coefficient between the fluorescence signal of FITC-loaded nanoparticles and the LysoTracker Red (PCC ± SD plot over time, n = 5). PCC analysis was performed by using MATLAB software. ® Drug release in vitro. Thanks to the therapeutic effectiveness towards a wide range of cancers (carcinomas, sarcomas and hematological cancers)58 and to its fluorescent properties59, doxorubicin (Dox) was selected as anticancer drug model for the current work. To be efficient, DDSs should guarantee a stable encapsulation of the drug, combined with a controlled release at the specific target. For bare MSNPs, the environmental pH plays a crucial role on the drug release kinetics. Further information about the mechanism of Dox uptake and release in/ from MSNPs is reported in SI (Supplementary Fig. S9). Gao et al. have shown that the release rate of Dox in vitro is accelerated at acidic pH, although a relatively smaller amount can be released at neutral pH as well60. In the particle design proposed here, in addition to confer to the MSNPs active targeting towards cancer cells and the capability to induce endosomal rupture, the HA-PEI polymeric bilayer will function as a capping agent, preventing the leakage of the drug before reaching the intracellular environment. At neutral pH, according to the pKa values of PEI and silica hydroxyl groups53,61, the electrostatic interactions guarantee a stable attachment of the PEI shell to the particles, impeding the discharge of Dox in blood circulation. At acidic pH, instead, as the majority of the hydroxyl groups of the silica particles are protonated, the electrostatic interactions are minimized, reducing the capping effect of the polymeric coating and facilitating the drug release in the cellular acidic compartments. In this context, Meng and co-workers reported that PEI coating does not hinder the Dox release at acidic pH47. Scientific Reports | (2019) 9:2666 | https://doi.org/10.1038/s41598-019-39107-3 6 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 5. Drug release in vitro and in cellulo. (a) Time-dependent in vitro release profile of Dox from HAPEIMSNPs_Dox (0–72 h) at pH 4.5 (red circle), pH 4.5 + Hyal-1 (red triangle), pH 6 (blue circle), pH 6 + Hyal-2 (blue triangle) and pH 7.4 (green triangle) (each point consists of mean ± SD, n = 3). (b–d) Fluorescence images of Dox released from HAPEI-MSNPs_Dox inside A549 cells after 3, 24 and 48 h (b-d, respectively). Dox channel (in red), DIC (gray) and merged images are shown from left to right, respectively. The contrast of the red channel was kept constant in all images. In order to evaluate the capping effect of the polymeric HA-PEI bilayer proposed here, the release kinetics of Dox from HAPEI-MSNPs was estimated in vitro. As depicted in Fig. 5a, functionalization of MSNPs with a HA-PEI bilayer resulted in particles with no drug release in both neutral (pH 7) and acidic environments (pH 6 and 4.5). This suggests that the stability of the shell is enhanced by the external HA layer, which likely hinders the polymer detachment, making the coating more compact and stable, even at acidic pH, thanks to the amide bond links to the HA. In the cellular environment, the external HA shell can be degraded by digestive intracellular enzymes, thereby promoting the discharge of the drug exclusively within the target cell. The main HA digestive enzymes are Hyaluronidase-1 (Hyal-1), which is normally located inside endosomes and lysosomes, and Hyaluronidase-2 (Hyal-2), mainly present on the plasma membrane62,63. While most degradation occurs in the acidic compartments, Hyal-2 can already degrade the high molecular weight HA into smaller fragments during the ligand-receptor binding, immediately prior to endocytosis63. Scientific Reports | (2019) 9:2666 | https://doi.org/10.1038/s41598-019-39107-3 7 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 6. Anticancer efficiency of Dox-loaded HAPEI-MSNPs. Viability tests of A549 cells incubated with HAPEI-MSNPs_Dox (wine red line column), free Dox (violet column) and empty HAPEI-MSNPs (light gray) for 72 h. Aliquots of 2, 4, 6, 8, 10 μL corresponding to final Dox concentrations of 80, 160, 240, 320, 400 nM and particle concentration of 20, 40, 60, 80, 100 μg/mL, respectively, were added to 1 mL of cell culture medium. All data are shown as mean ± SD (n = 3) with ns = (p > 0.05), *(p < 0.05), **(p < 0.01) and ***p < 0.001). Enzyme-mediated HA degradation and subsequent drug release was evaluated by incubating the Dox loaded HAPEI-MSNPs in different solutions at 37 °C. MES buffer (pH 6) with Hyal-2 was selected to mimic the extracellular matrix in tumor tissue, and acetate buffer (pH 4.5) containing Hyal-1 was used to simulate the late endosomes and lysosomes. The amount of Dox released at different incubation times (3, 12, 24, 48, 72 h) is shown in Fig. 5a. While in absence of enzymes and independently of the pH the percentage of Dox released was negligible, upon enzymatic digestion by Hyal-1 (pH 4.5) or Hyal-2 (pH 6), the release profiles were similar to those of bare MSNP (Supplementary Fig. S6). Similarly to bare MSNPs, Dox release kinetics were faster at more acidic pH, which is in agreement with previous reports43. The addition of Hayl-2 to the solution mimicking the extracellular matrix (pH 6) led to a total release of Dox from HAPEI-MSNPs of 58 ± 3% after 72 h. Notably, after only 3 h, 15% of the drug had been already released, suggesting that a partial digestion of HA on the plasma membrane can facilitate some Dox release. The addition of Hyal-1 at 4.5 pH (similar to the endo-/lysosomes) turned out to be the condition with the higher amount of Dox released, reaching a percentage of 68 ± 1% in 72 h. The Dox release profile from HAPEI-MSNPs in the presence of hyaluronidase demonstrates that only enzyme-mediated degradation of the polymeric coating, which occurs exclusively in the cellular environment, triggers drug release from the particles. Drug release in cellulo. In order to evaluate drug release kinetics in cellulo, HAPEI-MSNPs loaded with Dox were added to A549 cells and intracellular Dox release was monitored using fluorescence microscopy (Fig. 5b–d). When adding pure Dox to cells, fluorescence could be detected uniformly in the cytoplasmic region after 24 h, with no signal coming from the cell nucleus (Supplementary Fig. S7). The absence of fluorescence in the cell nucleus is associated to Dox intercalation between the DNA base pairs. As reported by several research groups, nuclear penetration causes a drastic quenching of Dox fluorescence64–66, up to 95% of its intrinsic emission67. Similar to the pure drug, after 3 h of incubation with Dox-loaded HAPEI-MSNPs, fluorescence could be detected in the cytoplasm of A549 cells. The weak dispersed signal in the cytoplasmic area was attributed to a small ratio of Dox release within the 3 h of incubation (which is in agreement with the results obtained in vitro in the presence of HA-degrading enzymes). While cells incubated with the pure drug only show a disperse fluorescence over the whole cytoplasmic region (Supplementary Fig. S7), when Dox-loaded nanoparticles are used, it was possible to observe bright dots in the intracellular environment (Fig. 5b). These bright dots were attributed to the HAPEI-MSNPs containing Dox. At longer time intervals (24 and 48 h), the fluorescence signal from Dox was more intense over the cytoplasm, while the bright spot-like signals arising from the particles became dimmer (Fig. 5c,d). This suggests that during time Dox was released from the particles into the intracellular environment (note that after 3 h the cells were washed, stopping further uptake of any drug and/or particles). This change in the distribution of Dox fluorescence signal was observed in all cells (Supplementary Fig. S8) and is in agreement with the enzyme-mediated release profile obtain in the in vitro experiments. Anticancer efficiency: cell viability tests. In order to evaluate the efficiency of the newly developed polymer-coated particles as anticancer DDSs, we monitored the cell viability 72 h after treatment with free Dox, Dox-loaded HAPEI-MSNPs and empty HAPEI-MSNPs, at different concentration of drug/particles (Fig. 6). While at low concentration (
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Running head: SWOT NALAYSIS OF INNOVATION

Eureka Moment-SWOT Analysis
Student
Institution
Date

1

SWOT NALAYSIS OF INNOVATION

2
A

1. Tracing the Scientific Method
Introduction of the Innovation
The article is about a strategy or an innovation that will help to internalize the present
nanoparticles to target cancer cells in the human body (Fortuni, Inose, Ricci, Fujita, Van
Zundert, Masuhara, & Uji-i, 2019). The report brings forth the observation, research question,
hypothesis, experiment, results and the conclusion which are critical to establishing the SWOT
analysis to the innovation.
Observations
In this research, the hyaluronic acid (HA) has demonstrated biocompatibility and does
not show any signs of harm which has made it get the approval of the FDA (Fortuni, et al.,
2019). Another observation in the experiment was that the Transmission electron microscopy
(TEM) images of uncoated MSNPs showed a uniform mesoporous frame and the particles as far
are size and shape are, concerned showed homogeneity without any aggregate observed.
Research Question
The research question in this study is as to whether polymeric engineering of
nanoparticles is highly efficient in multifunctional drug delivery systems.
Hypothesis
The developed DDS has all the ability to escape the endosomal pathway when the
polymeric is functionalized (Fortuni, et al., 2019). It is believed that the hyaluronic acid (H...


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