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Format:
</label>
<select id="email-citation-format" name="citation-format" class="action-panel-selector email-citation-format">
<option selected="selected" value="summary">Summary</option>
<option value="summary-text">Summary (text)</option>
<option value="abstract">Abstract</option>
<option value="abstract-text">Abstract (text)</option>
</select>
</div>
<div class="include-supplemental-container">
<input type="checkbox" aria-label="Include MeSH and other data" name="include-supplemental" id="email-include-supplemental" class="email-include-supplemental">
<label for="email-include-supplemental" class="email-include-supplemental-label">MeSH and other data</label>
</div>
<div class="form-field recaptcha ">
<div class="g-recaptcha" id="id-recaptcha" data-sitekey="6LfsWHMdAAAAAClKbtOpjQ2pMjgsGxvv7NdZW9uI"></div>
</div>
<div id="captcha-error-message" class="usa-input-error-message captcha-validation-message" role="alert"></div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="send">
Send email
</button>
<button class="action-panel-cancel"
aria-label="Close 'Email citations' panel"
ref="linksrc=close_email_panel"
aria-controls="email-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="cancel">
Cancel
</button>
</div>
<input type="hidden" name="email-search-details" value="" />
<input type="hidden" name="email-search-details-hash" value="0e42663a6c3bd85498fcb88798998fed7bfdc45d457db35281e41afe13cc0524" />
</form>
</div>
</div>
<div id="collections-action-panel"
class="collections-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-collections-open-panel-enabled="false"
data-collections-open-panel-url-hash="#open-collections-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to Collections
</h3>
<form id="collections-action-panel-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-myncbi-max-collection-name-length="100"
data-add-to-collection-max-amount="1000"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/">
<input type="hidden" name="csrfmiddlewaretoken" value="OxII2yaCYIo2tJSQtqEdgbCWNLRsmhtN5ksNFoDYyjbwnaYG6JhfKj8AlPzEfS17">
<div class="choice-group" role="radiogroup">
<ul class="radio-group-items">
<li>
<input type="radio"
id="collections-action-panel-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_new">
<label for="collections-action-panel-new">Create a new collection</label>
</li>
<li>
<input type="radio"
id="collections-action-panel-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_existing">
<label for="collections-action-panel-existing">Add to an existing collection</label>
</li>
</ul>
</div>
<div class="controls-wrapper">
<div class="action-panel-control-wrap new-collections-controls">
<label for="collections-action-panel-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label>
<input
type="text"
name="add-to-new-collection"
id="collections-action-panel-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="save_share"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection">
<div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div>
</div>
<div class="action-panel-control-wrap existing-collections-controls">
<label for="collections-action-panel-add-to-existing" class="action-panel-label">
Choose a collection:
</label>
<select id="collections-action-panel-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="save_share"
data-ga-action="select_collection"
data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'">
</select>
<div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br>
<a href="#">Please try again</a>
</div>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="add">
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to Collections' panel"
ref="linksrc=close_collections_panel"
aria-controls="collections-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="bibliography-action-panel"
class="bibliography-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-bibliography-open-panel-enabled="false"
data-bibliography-open-panel-url-hash="#open-bibliography-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to My Bibliography
</h3>
<form id="bibliography-action-panel-form"
class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-add-to-bibliography-max-amount="100"
data-add-to-bibliography-batch-size="10"
data-bibliography-delegates-url="/list-bibliography-delegates/"
data-add-to-bibliography-url="/add-to-bibliography/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/">
<input type="hidden" name="csrfmiddlewaretoken" value="OxII2yaCYIo2tJSQtqEdgbCWNLRsmhtN5ksNFoDYyjbwnaYG6JhfKj8AlPzEfS17">
<div class="action-panel-control-wrap bibliographies-controls">
<div class="choice-group">
<ul class="bibliographies-action-add radio-group-items">
<li>
<input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/>
<label for="my-bibliography">My Bibliography</label>
</li>
</ul>
</div>
</div>
<div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your delegates due to an error<br>
<a href="#">Please try again</a>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore>
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to bibliography' panel"
ref="linksrc=close_bibliography_panel"
aria-controls="bibliography-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="mybib"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true"
data-saved-search-open-panel-enabled="false"
data-saved-search-open-panel-url-hash="#open-saved-search-panel">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your saved search
</h2>
<form id="saved-search-action-panel-form"
class="saved-search-action-panel-form action-panel-content action-form"
data-create-saved-search-url="/create-saved-search/"
data-try-search-terms-url="/try-search-term/"
data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/">
<input type="hidden" name="csrfmiddlewaretoken" value="OxII2yaCYIo2tJSQtqEdgbCWNLRsmhtN5ksNFoDYyjbwnaYG6JhfKj8AlPzEfS17">
<div class="action-panel-control-wrap">
<label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk">
Name of saved search:
</label>
<input maxlength="200"
type="text"
name="saved-search-name"
id="saved-search-name"
class="saved-search-name"
value=""
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-term" class="action-panel-label required-field-asterisk">
Search terms:
</label>
<textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea>
</div>
<div class="test-search-term-wrap">
<a href="#" class="try-search-term">Test search terms</a>
</div>
<div class="choice-group action-panel-extra-margin-top">
<span class="action-panel-label" id="fieldset-label">
Would you like email updates of new search results?
</span>
<fieldset id="saved-search-alert" aria-describedby="fieldset-label">
<legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend>
<ul class="radio-group-items">
<li>
<input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked>
<label for="saved-search-alert-yes" class="action-panel-label">Yes</label>
</li>
<li>
<input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no">
<label for="saved-search-alert-no" class="action-panel-label">No</label>
</li>
</ul>
</fieldset>
</div>
<div class="alert-schedule-wrap">
<div class="action-panel-control-wrap">
<label class="action-panel-label">
Email:
</label>
<span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span>
</div>
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="saved-search-frequency" class="action-panel-label">
Frequency:
</label>
<select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency">
<option value="monthly">Monthly</option>
<option value="weekly">Weekly</option>
<option value="daily">Daily</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-monthly-additional">
<label for="saved-search-monthly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-monthly-on-day" class="no-border-panel-selector">
<option value="Sunday">The first Sunday</option>
<option value="Monday">The first Monday</option>
<option value="Tuesday">The first Tuesday</option>
<option value="Wednesday">The first Wednesday</option>
<option value="Thursday">The first Thursday</option>
<option value="Friday">The first Friday</option>
<option value="Saturday">The first Saturday</option>
<option value="day">The first day</option>
<option value="weekday">The first weekday</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-weekly-additional">
<label for="saved-search-weekly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day">
<option value="Sunday">Sunday</option>
<option value="Monday">Monday</option>
<option value="Tuesday">Tuesday</option>
<option value="Wednesday">Wednesday</option>
<option value="Thursday">Thursday</option>
<option value="Friday">Friday</option>
<option value="Saturday">Saturday</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-report" class="action-panel-label">
Report format:
</label>
<select id="saved-search-report" class="no-border-panel-selector saved-search-report">
<option value="DocSum">Summary</option>
<option value="DocSumText">Summary (text)</option>
<option value="Abstract">Abstract</option>
<option value="AbstractText">Abstract (text)</option>
<option value="MEDLINE">PubMed</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-amount" class="action-panel-label">
Send at most:
</label>
<select id="saved-search-amount" class="no-border-panel-selector saved-search-amount">
<option value="1">1 item</option>
<option value="5" selected>5 items</option>
<option value="10">10 items</option>
<option value="20">20 items</option>
<option value="50">50 items</option>
<option value="100">100 items</option>
<option value="200">200 items</option>
</select>
</div>
<div>
<input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result">
<label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox">
Send even when there aren't any new results
</label>
</div>
<div class="action-panel-control-wrap option-text-in-email-wrap">
<label for="saved-search-email-text" class="action-panel-label">
Optional text in email:
</label>
<textarea name="saved-search-email-text"
id="saved-search-email-text"
class="saved-search-email-text"></textarea>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Saving..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Save
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your saved search' panel"
ref="linksrc=close_saved_search_panel"
aria-controls="saved-search-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Create a file for external citation management software
</h2>
<form id="citation-manager-action-panel-form"
class="action-panel-content action-form"
action="/results-export-ids/"
data-by-search-action="/results-export-search-data/"
data-by-ids-action="/results-export-ids/"
method="post"
data-by-search-method="post"
data-by-ids-method="post">
<input type="hidden" name="csrfmiddlewaretoken" value="OxII2yaCYIo2tJSQtqEdgbCWNLRsmhtN5ksNFoDYyjbwnaYG6JhfKj8AlPzEfS17">
<input name="results-format" type="hidden" value="pubmed"/>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="save">
Create file
</button>
<button class="action-panel-cancel"
aria-label="Close 'Send citations to citation manager' panel"
ref="linksrc=close_citation_manager_panel"
aria-controls="citation-manager-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your RSS Feed
</h2>
<form id="rss-action-panel-form"
class="rss-action-panel-form action-panel-content action-form"
data-create-rss-feed-url="/create-rss-feed-url/"
data-search-form-term-value="">
<input type="hidden" name="csrfmiddlewaretoken" value="OxII2yaCYIo2tJSQtqEdgbCWNLRsmhtN5ksNFoDYyjbwnaYG6JhfKj8AlPzEfS17">
<div class="action-panel-control-wrap">
<label for="rss-name" class="action-panel-label required-field-asterisk">
Name of RSS Feed:
</label>
<input maxlength="200"
placeholder="Name"
type="text"
name="rss-name"
id="rss-name"
class="rss-name"
value=''
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="rss-limit-wrap">
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="rss-limit" class="action-panel-label">
Number of items displayed:
</label>
<select id="rss-limit" class="no-border-panel-selector rss-limit">
<option value="5">5</option>
<option value="10">10</option>
<option value="15" selected="selected">15</option>
<option value="20">20</option>
<option value="50">50</option>
<option value="100">100</option>
</select>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Creating..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Create RSS
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your RSS' panel"
ref="linksrc=close_rss_panel"
aria-controls="rss-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
<div class="action-panel-control-wrap rss-link-copy-wrap">
<label for="rss-link" class="usa-sr-only">RSS Link</label>
<input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link">
<button
type="button"
disabled
class="rss-link-copy-button disabled"
data-ga-category="save_share"
data-ga-action="rss"
data-ga-label="copy">
Copy
</button>
</div>
</form>
</div>
</div>
</div>
</div>
<div class="article-page" id="article-page" data-article-pmid="38872047">
<aside class="page-sidebar">
<div class="inner-wrap">
<div class="full-text-links">
<div class="full-view">
<h3 class="title">
Full text links
</h3>
<div class="full-text-links-list">
<a class="link-item
dialog-focus"
href="https://dx.doi.org/10.1007/s13346-024-01644-4"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=False&amp;PrId=3055&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=38872047&amp;db=pubmed&amp;nlmid=101540061"
title="See full text options at Springer"
data-ga-category="full_text"
data-ga-action="Springer"
data-ga-label="38872047"
><img src="https://cdn.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--production.springer.de-OnlineResources-Logos-springerlink.gif" alt="Springer full text link"><span class="text">
Springer
</span></a><a class="link-item
pmc
"
href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/38872047/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=38872047&amp;db=pubmed&amp;nlmid=101540061"
title="Free full text at PubMed Central"
data-ga-category="full_text"
data-ga-action="PMC"
data-ga-label="38872047"
><span class="text">
Free PMC article
</span></a>
</div>
</div>
<div class="short-view">
<a href="#" class="full-text-links-button full-text-links-dialog-trigger">
Full text links
</a>
</div>
</div>
<div class="actions-buttons sidebar"><h3 class="title">Actions</h3><div class="inner-wrap"><button class="citation-button citation-dialog-trigger"
aria-label="Open dialog with citation text in different styles"
data-ga-category="save_share"
data-ga-action="cite"
data-ga-label="open"
data-all-citations-url="/38872047/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/38872047/export/"><span class="button-label">Cite</span></button><link type="text/css" href="ncbi-overlay-block/src/overlay-block.css"><div class="collections-button-container" data-article-id="38872047" data-article-db="pubmed"><button class="collections-button collections-dialog-trigger"
aria-label="Save article in MyNCBI collections."
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_button"
data-collections-open-dialog-enabled="false"
data-collections-open-dialog-url="https://account.ncbi.nlm.nih.gov/?back_url=https%3A%2F%2Fpubmed.ncbi.nlm.nih.gov%2F38872047%2F%23open-collections-dialog"
data-in-collections="false"><span class="button-label">Collections</span></button><div class="overlay" role="dialog"><div id="collections-action-dialog"
class="dialog collections-dialog"
aria-hidden="true"><div class="title">Add to Collections</div><div class="collections-action-panel action-panel"><form id="collections-action-dialog-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-myncbi-max-collection-name-length="100"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/"><input type="hidden" name="csrfmiddlewaretoken" value="OxII2yaCYIo2tJSQtqEdgbCWNLRsmhtN5ksNFoDYyjbwnaYG6JhfKj8AlPzEfS17"><div class="choice-group" role="radiogroup"><ul class="radio-group-items"><li><input type="radio"
id="collections-action-dialog-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_new"><label for="collections-action-dialog-new">Create a new collection</label></li><li><input type="radio"
id="collections-action-dialog-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_existing"><label for="collections-action-dialog-existing">Add to an existing collection</label></li></ul></div><div class="controls-wrapper"><div class="action-panel-control-wrap new-collections-controls"><label for="collections-action-dialog-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label><input
type="text"
name="add-to-new-collection"
id="collections-action-dialog-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="collections_button"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection"><div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div></div><div class="action-panel-control-wrap existing-collections-controls"><label for="collections-action-dialog-add-to-existing" class="action-panel-label">
Choose a collection:
</label><select id="collections-action-dialog-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
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Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy
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</a><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institute of Biomedical Technologies (ITB), Center for Biomedical Research of the Canary Islands (CIBICAN), Universidad de La Laguna, La Laguna, 38200, Spain." href="#short-view-affiliation-2" ref="linksrc=author_aff">
2
</a></sup><span class="comma">,&nbsp;</span></span><span class="authors-list-item "><a class="full-name"
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data-ga-label="Patricia García-García">Patricia García-García</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Department of Chemical Engineering and Pharmaceutical Technology, Universidad de La Laguna, La Laguna, 38200, Spain. pgarciag@ull.edu.es." href="#short-view-affiliation-4" ref="linksrc=author_aff">
4
</a><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institute of Biomedical Technologies (ITB), Center for Biomedical Research of the Canary Islands (CIBICAN), Universidad de La Laguna, La Laguna, 38200, Spain. pgarciag@ull.edu.es." href="#short-view-affiliation-5" ref="linksrc=author_aff">
5
</a></sup><span class="comma">,&nbsp;</span></span><span class="authors-list-item "><a class="full-name"
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data-ga-label="Araceli Delgado">Araceli Delgado</a><sup class="affiliation-links"><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Department of Chemical Engineering and Pharmaceutical Technology, Universidad de La Laguna, La Laguna, 38200, Spain. adelgado@ull.edu.es." href="#short-view-affiliation-6" ref="linksrc=author_aff">
6
</a><span class="author-sup-separator">&nbsp;</span><a class="affiliation-link" title="Institute of Biomedical Technologies (ITB), Center for Biomedical Research of the Canary Islands (CIBICAN), Universidad de La Laguna, La Laguna, 38200, Spain. adelgado@ull.edu.es." href="#short-view-affiliation-7" ref="linksrc=author_aff">
7
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<li data-affiliation-id="short-view-affiliation-1"
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><sup class="key">1</sup> Department of Chemical Engineering and Pharmaceutical Technology, Universidad de La Laguna, La Laguna, 38200, Spain.</li>
<li data-affiliation-id="short-view-affiliation-2"
id="short-view-affiliation-2"
><sup class="key">2</sup> Institute of Biomedical Technologies (ITB), Center for Biomedical Research of the Canary Islands (CIBICAN), Universidad de La Laguna, La Laguna, 38200, Spain.</li>
<li data-affiliation-id="short-view-affiliation-3"
id="short-view-affiliation-3"
><sup class="key">3</sup> Department of Basic Medical Sciences, Universidad de La Laguna, La Laguna, 38200, Spain.</li>
<li data-affiliation-id="short-view-affiliation-4"
id="short-view-affiliation-4"
><sup class="key">4</sup> Department of Chemical Engineering and Pharmaceutical Technology, Universidad de La Laguna, La Laguna, 38200, Spain. pgarciag@ull.edu.es.</li>
<li data-affiliation-id="short-view-affiliation-5"
id="short-view-affiliation-5"
><sup class="key">5</sup> Institute of Biomedical Technologies (ITB), Center for Biomedical Research of the Canary Islands (CIBICAN), Universidad de La Laguna, La Laguna, 38200, Spain. pgarciag@ull.edu.es.</li>
<li data-affiliation-id="short-view-affiliation-6"
id="short-view-affiliation-6"
><sup class="key">6</sup> Department of Chemical Engineering and Pharmaceutical Technology, Universidad de La Laguna, La Laguna, 38200, Spain. adelgado@ull.edu.es.</li>
<li data-affiliation-id="short-view-affiliation-7"
id="short-view-affiliation-7"
><sup class="key">7</sup> Institute of Biomedical Technologies (ITB), Center for Biomedical Research of the Canary Islands (CIBICAN), Universidad de La Laguna, La Laguna, 38200, Spain. adelgado@ull.edu.es.</li>
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Abstract
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<p>
Gene therapy holds significant promise as a therapeutic approach for addressing a diverse range of diseases through the suppression of overexpressed proteins and the restoration of impaired cell functions. Developing a nanocarrier that can efficiently load and release genetic material into cells remains a challenge. The primary goal of this study is to develop formulations aimed to enhance the therapeutic potential of GapmeRs through technological approaches. To this end, lipid-polymeric hybrid nanoparticles (LPHNPs) with PLGA, DC-cholesterol, and DOPE-mPEG<sub>2000</sub> were produced by conventional single-step nanoprecipitation (SSN) and microfluidic (MF) methods. The optimized nanoparticles by SSN have a size of 149.9 ± 18.07 nm, a polydispersity index (PdI) of 0.23 ± 0.02, and a zeta potential of (ZP) of 29.34 ± 2.44 mV, while by MF the size was 179.8 ± 6.3, a PdI of 0.24 ± 0.01, and a ZP of 32.25 ± 1.36 mV. Furthermore, LPHNPs prepared with GapmeR-protamine by both methods exhibit a high encapsulation efficiency of approximately 90%. The encapsulated GapmeR is completely released in 24 h. The LPHNP suspensions are stable for up to 6 h in 10% FBS at pH 5.4 and 7.4. By contrast, LPHNPs remain stable in suspension in 4.5% albumin at pH 7.4 for 24 h. Additionally, LPHNPs were successfully freeze-dried using trehalose in the range of 2.5-5% as cryoprotectant The LPHNPs produced by MF and SSN increase, 6 and 12 fold respectively, GapmeR cell uptake, and both of them reduce by 60-70% expression of Tob1 in 48 h.Our study demonstrates the efficacy of the developed LPHNPs as carriers for oligonucleotide delivery, offering valuable insights for their scale up production from a conventional bulk methodology to a high-throughput microfluidic technology.
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Endosomal escape; Gene therapy; Lipid-polymeric hybrid nanoparticles; Microfluidics; Nanoparticles stability.
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<p xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:p1="http://pubmed.gov/pub-one">Declarations. Ethics approval: Not applicable. Competing interests: The authors have non-financial interests to disclose.</p>
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<div class="figure-caption-contents"><p> Enlarged perspective of the micromixer chip captured with stereomicroscope at 7.8 magnifications </p></div>
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<div class="figure-caption-contents"><p> Physicochemical characteristics of LPHNPs elaborated by conventional bulk single-step nanoprecipitation (SSN) using DOPE-mPEG… </p></div>
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<div class="figure-caption-contents">Physicochemical characteristics of LPHNPs elaborated by conventional bulk single-step nanoprecipitation (SSN) using DOPE-mPEG<sub>2000</sub> and DC-Chol (molar ratio 1:3) with protamine as a complexing agent. (<b>A</b>) Average size (bars) and ZP (symbol) and (<b>B</b>) Polydispersion index (PdI). Dashed line indicated values of 200 nm and PdI=0.3 in the respective graph and dotted line 0 mV. <i>n</i>=3</div>
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<div class="figure-caption-contents"><p> Physicochemical characteristics of LPHNPs elaborated… </p></div>
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<div class="figure-caption-contents"><p> Physicochemical characteristics of LPHNPs elaborated by microfluidic (MF) using DOPE-mPEG <sub> 2000 </sub> and DC-Chol… </p></div>
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<div class="figure-caption-contents">Physicochemical characteristics of LPHNPs elaborated by microfluidic (MF) using DOPE-mPEG<sub>2000</sub> and DC-Chol (molar ratio 1:3) with protamine as a complexing agent. Flow Rate Ratio (FRR) of 3 and Total Flow Rate (TFR) of 0.8 mL/min. (<b>A</b>) Average size (bars) and ZP (symbol) and (<b>B</b>) Polydispersion index (PdI). Dashed line indicated values of 200 nm and PdI=0.3 in the respective graph and dotted line 0 mV. <i>n</i>=3</div>
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<div class="figure-caption-contents"><p> Images from LPHNPs made by… </p></div>
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<div class="figure-caption-contents"><p> Images from LPHNPs made by ( <b> A </b> ) conventional single-step nanoprecipitation (SSN) and… </p></div>
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<div class="figure-caption-contents">Images from LPHNPs made by (<b>A</b>) conventional single-step nanoprecipitation (SSN) and (<b>B</b>) microfluidics (MF)</div>
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<div class="figure-caption-contents"><p> GapmeR release profile from LPHNPs… </p></div>
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<p> Fig. 5 </p>
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<div class="figure-caption-contents"><p> GapmeR release profile from LPHNPs ( <b> A </b> ) at different pHs, 7.4 and… </p></div>
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<div class="figure-caption-contents">GapmeR release profile from LPHNPs (<b>A</b>) at different pHs, 7.4 and 5.5 in 10% FBS (<b>B</b>) at pH=7.4 for the first two hours and continued at pH=5.5 until the end of the assay. Continuous line and dotted line represent LPHNPs developed by SSN and MF, respectively. 37˚C and 300 rpm, <i>n</i>=3</div>
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<div class="figure-caption-contents">(<b>A</b>) Sizes and (<b>B</b>) PdI of LPHNPs in different stabilizers: 0.9% NaCl, 2.5% trehalose and 10% trehalose at time 0 and 24 h at 4 °C. Filled bars and striped bars represent LPHNPs developed by SSN and MF, respectively. Symbols denote statistically significant differences with native LPHNPs at time zero, prepared by SSN (#) and prepared by MF (*). <i>n</i>=3, <i>p</i>&lt;0.05</div>
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<p> Fig. 7 </p>
</strong>
<div class="figure-caption-contents"><p> Increase in LPHNPs size elaborated… </p></div>
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<p> Fig. 7 </p>
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<div class="figure-caption-contents"><p> Increase in LPHNPs size elaborated by SSN and MF ( <b> A </b> ) in… </p></div>
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Fig. 7
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<div class="figure-caption-contents">Increase in LPHNPs size elaborated by SSN and MF (<b>A</b>) in 10% FBS at different pHs, 7.4, 5.5, and 7.4 during the first 2 h and 5.5 during the rest of the assay (<b>B</b>) in 4.5% BSA at pH 7.4. Filled bars and striped bars represent LPHNPs developed by SSN and MF, respectively. Symbols denote statistically significant differences with native LPHNPs at time zero prepared by SSN (#) and prepared by MF (*). <i>n</i>=3, <i>p</i>&lt;0.05</div>
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<p> Fig. 8 </p>
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<div class="figure-caption-contents"><p> ( <b> A </b> ) Sizes and… </p></div>
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<p> Fig. 8 </p>
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<div class="figure-caption-contents"><p> ( <b> A </b> ) Sizes and ( <b> B </b> ) PdI of LPHNPs dispersed in… </p></div>
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Fig. 8
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<div class="figure-caption-contents">(<b>A</b>) Sizes and (<b>B</b>) PdI of LPHNPs dispersed in different concentrations of trehalose before and after being freeze-dried and resuspended in 1 ml of MilliQ water. Filled bars and striped bars represent LPHNPs developed by SSN and MF, respectively. Symbols denote statistically significant differences with native LPHNPs at time zero, prepared by SSN (#) and prepared by MF (*). <i>n</i>=3, <i>p</i>&lt;0.05</div>
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<div class="figure-caption-contents"><p> ( <b> A </b> - <b> L </b> ) Representative C2C12 cells exposed to naked GapmeR-FAM (… </p></div>
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Fig. 9
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<div class="figure-caption-contents">(<b>A</b>-<b>L</b>) Representative C2C12 cells exposed to naked GapmeR-FAM (<b>A</b>-<b>D</b>); (<b>E</b>-<b>H</b>) LPHNPs loaded with GapmeR-FAM elaborated by bulk SSN; (<b>I</b>-<b>L</b>) LPHNPs loaded with GapmeR-FAM elaborated by MF. Each representative confocal image is split into four sections: green channel representing FAM-GapmeR signal; red channel representing the late endosomal-lysosomal compartment labeled with the biomarker Lysotracker Red; “Merge” shows the green, red and blue (nuclei labeled with DAPI) signals; “Overlap” represents the colocalization overlays revealing as white dots those areas with GapmeR-FAM and Lysotracker Red positive signals. “Overlap” representations were obtained using the BIOP JACoP. (<b>M</b>) Quantification of the internalization ratio of GapmeR-FAM in cells exposed to LPHNPs prepared by bulk SSN or MF was calculated as the mean number of cytoplasmic FAM-positive foci per cell normalized to cells exposed to naked GapmeR-FAM (free). (<b>N</b>) Quantification using the BIOP JACoP plugin from FIJI of the percentage of GapmeR-FAM area that localizes within the endolysosomal Lysotracker-positive compartment (green-positive/red-positive “Overlap” area, colored bars) or that is distributed through the cytoplasm (green-positive/red-negative area, white bars) relativized to the total GapmeR-FAM area (total green-positive area, 100% bar). (<b>O</b>) Analysis of relative <i>Tob1</i> mRNA fold levels 48 h after treatment of C2C12 cells with either control (white bars) or anti-<i>Tob1</i> GapmeRs (colored bars) delivered by Dharmafect-1 transfection (DF) or HLPNPs elaborated by bulk SSN or MF as indicated. Scale bar: 10 μm (<b>A</b>-<b>L</b>); (*, **, ***) denotes statistically significant differences (<i>p</i>&lt;0.05, <i>p</i>&lt;0.005, <i>p</i>&lt;0.0005) with the naked GapmeR-FAM or among bulk SSN and MF, as indicated</div>
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