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Biocompatible near-infrared quantum dots delivered to the skin by microneedle patches record vaccination

Authors:
  • Institute of Chemistry, Chinese Academy of Sciences

Abstract and Figures

Accurate medical recordkeeping is a major challenge in many low-resource settings where well-maintained centralized databases do not exist, contributing to 1.5 million vaccine-preventable deaths annually. Here, we present an approach to encode medical history on a patient using the spatial distribution of biocompatible, near-infrared quantum dots (NIR QDs) in the dermis. QDs are invisible to the naked eye yet detectable when exposed to NIR light. QDs with a copper indium selenide core and aluminum-doped zinc sulfide shell were tuned to emit in the NIR spectrum by controlling stoichiometry and shelling time. The formulation showing the greatest resistance to photobleaching after simulated sunlight exposure (5-year equivalence) through pigmented human skin was encapsulated in microparticles for use in vivo. In parallel, microneedle geometry was optimized in silico and validated ex vivo using porcine and synthetic human skin. QD-containing microparticles were then embedded in dissolvable microneedles and administered to rats with or without a vaccine. Longitudinal in vivo imaging using a smartphone adapted to detect NIR light demonstrated that microneedle-delivered QD patterns remained bright and could be accurately identified using a machine learning algorithm 9 months after application. In addition, codelivery with inactivated poliovirus vaccine produced neutralizing antibody titers above the threshold considered protective. These findings suggest that intradermal QDs can be used to reliably encode information and can be delivered with a vaccine, which may be particularly valuable in the developing world and open up new avenues for decentralized data storage and biosensing.
Platform schematic and fluorescent probe characterization. (A) Fluorescent microparticles are distributed through an array of dissolvable microneedles in a distinct spatial pattern. (B) Microneedles are then applied to the skin for 2 to 5 min, resulting in dissolution of the microneedle matrix and retention of fluorescent microparticles. (C) A NIR LED and adapted smartphone are used to image patterns of fluorescent microparticles retained within the skin. By selectively embedding microparticles within microneedles used to deliver a vaccine, the resulting pattern of fluorescence detected in the skin can be used as an on-patient record of an individual's vaccination history. (D) Rapid photobleaching of organic dyes covered with pigmented human skin under simulated solar light. (E) Emission profiles of QDs (solid lines) show a blue shift with increased shelling time. Dashed line depicts absorption by the 5-hour shelling sample. Arrows indicate the relevant y axis for absorption (left) and emission (right). a.u., arbitrary units. (F) PL QY as a function of shelling time under different excitation wavelengths. (G) Relative photoluminescence intensity comparison of QDs with the commercial inorganic dyes IRDC2 and IRDC3 (blue bars) and corresponding emission peaks (empty purple circles and square). (H) Photostability of QDs covered with pigmented human skin under simulated solar light. (I) Transmission electron microscopy (TEM) and high-resolution TEM (inset) showing the size and crystal structure of ZnS:Al-coated CuInSe 2 QDs. Scale bars, 20 and 5 nm, respectively. n = 3 for all graphs containing error bars. Error bars indicate SD.
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Smartphone modifications and NIR marking detection in skin. (A) Photograph of disassembled LED used for NIR illumination at 780 nm combined with an 800-nm short-pass filter and aspheric condenser. (B) Photograph of disassembled NIR imaging smartphone consisting of a Google Nexus 5X smartphone with the internal short-pass filter removed and replaced with two external 850-nm long-pass filters set in a 3D-printed phone case. Images of a 16-needle microneedle patch containing PMMA-encapsulated QDs were collected with the adapted smartphone under ambient indoor lighting (C) without the 850-nm long-pass filters and (D) with the pair of 850-nm long-pass filters under LED illumination from the same distance. Inset shows an image at a higher exposure. (E) Optical and (F) SEM images of fluorescent microparticle-loaded microneedles before skin application. (G) Optical and (H) SEM images of microneedles after administration to explanted pig skin. Adapted smartphone images of pig skin before microneedle application (I) without and (J) with 850-nm long-pass filters. Adapted smartphone images of pig skin after application (K) without and (L) with 850-nm long-pass filters. Adapted smartphone images of pigmented human skin before microneedle application (M) without and (N) with the 850-nm longpass filters. Smartphone images of human skin after application (O) without and (P) with the 850-nm long-pass filters. Note: Scale bars in NIR-filtered images are approximate with (J), (L), (N), and (P) taken at about the same distance. Components in (A) and (B) cropped for clarity.
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McHugh et al., Sci. Transl. Med. 11, eaay7162 (2019) 18 December 2019
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BIOENGINEERING
Biocompatible near-infrared quantum dots delivered
to the skin by microneedle patches record vaccination
Kevin J. McHugh1*, Lihong Jing1,2*, Sean Y. Severt1, Mache Cruz1, Morteza Sarmadi1,3,
Hapuarachchige Surangi N. Jayawardena1‡, Collin F. Perkinson4, Fridrik Larusson5,
Sviatlana Rose1, Stephanie Tomasic1, Tyler Graf1, Stephany Y. Tzeng, James L. Sugarman1,
Daniel Vlasic6, Matthew Peters5, Nels Peterson5, Lowell Wood5, Wen Tang1, Jihyeon Yeom1,
Joe Collins1, Philip A. Welkhoff7, Ari Karchin5, Megan Tse1, Mingyuan Gao2, Moungi G. Bawendi4,
Robert Langer1||, Ana Jaklenec1||
Accurate medical recordkeeping is a major challenge in many low-resource settings where well-maintained
centralized databases do not exist, contributing to 1.5 million vaccine-preventable deaths annually. Here, we
present an approach to encode medical history on a patient using the spatial distribution of biocompatible,
near-infrared quantum dots (NIR QDs) in the dermis. QDs are invisible to the naked eye yet detectable when
exposed to NIR light. QDs with a copper indium selenide core and aluminum-doped zinc sulfide shell were tuned
to emit in the NIR spectrum by controlling stoichiometry and shelling time. The formulation showing the greatest
resistance to photobleaching after simulated sunlight exposure (5-year equivalence) through pigmented human
skin was encapsulated in microparticles for use in vivo. In parallel, microneedle geometry was optimized in silico
and validated ex vivo using porcine and synthetic human skin. QD-containing microparticles were then embedded
in dissolvable microneedles and administered to rats with or without a vaccine. Longitudinal in vivo imaging
using a smartphone adapted to detect NIR light demonstrated that microneedle-delivered QD patterns remained
bright and could be accurately identified using a machine learning algorithm 9 months after application. In ad-
dition, codelivery with inactivated poliovirus vaccine produced neutralizing antibody titers above the threshold
considered protective. These findings suggest that intradermal QDs can be used to reliably encode information
and can be delivered with a vaccine, which may be particularly valuable in the developing world and open up new
avenues for decentralized data storage and biosensing.
INTRODUCTION
Vaccines are exceptionally safe and effective, saving an estimated
2 million to 3 million lives annually (1). However, each year, 1.5 mil-
lion vaccine-preventable deaths occur due to undervaccination—
primarily in areas of the developing world with poor health care
infrastructure (2). One key barrier to improving vaccination cover-
age in these regions is the inability to accurately identify the immu-
nization status of infants given resource constraints, which can affect
the quality of care provided (3,4). These areas often lack accurate
medical recordkeeping systems and rely on vaccination campaigns
to distribute vaccines. However, investigations in response to recent
outbreaks of measles and mumps in the United States (5), Australia
(6), and Italy (7) have highlighted that poor immunization record-
keeping is not unique to developing nations.
Paper vaccination cards or certificates are the most widely used
records in the developing world but are subject to error (8) and pos-
sessed by only 60% of all households in low- and middle-income
countries (9). Without accurate vaccination records, health care
professionals lack the data needed to make informed decisions about
administering vaccines, often relying on parental recall (10). This may
result in the application of additional, unnecessary vaccine doses
and therefore undue cost or, more problematically, missed oppor-
tunities to vaccinate, which leave the child at risk for contracting
infectious diseases (8,10). As many as two-thirds of opportunities
to vaccinate may be missed in some areas (11), leading to a potential
30% drop in vaccination coverage (3). Several solutions have been
proposed including smartphone-based database applications (12),
fingerprinting (13), and near-field communication chips (14); how-
ever, these more technologically advanced methods have yet to
achieve widespread adoption due to difficulty of implementation.
Therefore, we aimed to develop a platform that is robust, inex-
pensive, and easy to use to overcome the primary obstacles to
implementation in the developing world. We hypothesized that
information, such as vaccination history, could be encoded invisibly
in the skin by applying a distinct pattern of near-infrared (NIR)
fluorescent microparticles using a microneedle patch. By providing
all of the requisite information on the patients themselves and
delivering microparticles in the same microneedles as the vaccine,
this platform offers several key advantages compared to traditional
1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology,
77 Massachusetts Avenue, Cambridge, MA 02139, USA. 2Key Laboratory of Colloid,
Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy
of Sciences, Bei Yi Jie 2, Zhong Guan Cun, Beijing 100190, China. 3Department of
Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts
Avenue, Cambridge, MA 02139, USA. 4Department of Chemistry, Massachusetts
Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
5Global Good, Intellectual Ventures Laboratory, 14360 SE Eastgate Way, Bellevue,
WA 98007, USA. 6Independent consultant, 119 Kendall Rd, Lexington, MA 02421,
USA (https://people.csail.mit.edu/drdaniel/). 7Institute for Disease Modeling, 3150
139th Ave. SE, Bellevue, WA 98005, USA.
*These authors contributed equally to this work.
†Present address: Department of Bioengineering, Rice University, Houston, TX
77005, USA.
‡Present address: Department of Chemistry, University of Alabama in Huntsville,
Huntsville, AL 35899, USA.
§Present address: School of Medicine, Johns Hopkins University, 733 N. Broadway,
Baltimore, MD 21205, USA.
||Corresponding author. Email: rlanger@mit.edu (R.L.); jaklenec@mit.edu (A.J.)
Copyright © 2019
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim
to original U.S.
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paper or electronic medical records including the following: (i) the
elimination of reliance on widely accessible, yet secure database of
patient information; (ii) lack of reliance on accurate patient identi-
fication and data entry by medical professionals; (iii) ability to make
rapid determinations of vaccination status; (iv) elimination of vac-
cination fraud; (v) capacity for population-level assessment of vac-
cine coverage; and (vi) minimal cost that is feasible to implement
in low-resource settings. By using a microneedle form factor, this
platform should easily assimilate into the future vaccination land-
scape because microneedles are currently in development for sev-
eral vaccines (15) and have shown advantages such as antigen
dose sparing, improved antigen stability, and ease of (self-)adminis-
tration compared to traditional soluble injections (1618). Mi-
croneedle patches for vaccination recordkeeping may not require
cold chain storage and could potentially have long shelf life, which
could greatly enhance feasibility of implementation in low-resource
settings.
RESULTS
Commercial dye and custom quantum dot characterization
To create a microneedle platform that could be applied to the skin,
rapidly dissolve, and leave behind particles that can later be imaged
to determine vaccination status (Fig.1,AtoC), we first needed to
identify a suitable candidate for long-term detection. We began by
investigating the use of commercial fluorophores with emission in
the NIR spectrum (fig. S1). To examine resistance to photobleach-
ing, a key criterion for use in our application, fluorescent dyes were
covered with pigmented cadaveric human skin and exposed to light
simulating the solar spectrum at sevenfold higher intensity. Despite
being heavily protected from light by overlying human skin, organ-
ic fluorophores photobleached within a few weeks of simulated sun
exposure (Fig.1D). Alternatively, inorganic commercial dyes demon-
strated considerable resistance to photobleaching but exhibited low
fluorescence intensity per mass and were difficult to process because
of their insolubility in both water and organic solvents. We then pur-
sued the use of colloidal quantum dots (QDs), also known as semi-
conductor nanocrystals, as potential fluorescent probes due to their
favorable brightness and photostability. We synthesized and char-
acterized more than 60 distinct combinations of copper indium
selenide cores and ZnS:Al shells (table S1). By changing the core
stoichiometry and shell thickness, we could control the peak emis-
sion wavelength (Fig.1E), enhance the photoluminescence quantum
yield (PL QY) (Fig.1F), and affect other optical properties (figs. S2
and S3 and table S2). For example, in one QD formulation, per-
forming the shelling process for 5 hours resulted in an increase
PL QY from 16.2 to 43.6% and a blue shift in the emission peak
from 964 to 891nm.
We then selected five QD formulations with emission peaks
ranging from 828 to 891nm to optimize invivo light transmission
and detection (Fig.1G). These QDs were exposed to simulated solar
light through pigmented cadaveric skin at sevenfold the intensity of
the sun for a period that simulated 5 years of day/night exposure.
One QD formulation, S10C5H, demonstrated substantially greater
resistance to photobleaching than other formulations (P<0.05),
retaining 13±3% of signal after five simulated years compared to
the next best candidate, which retained only 4±2% of its initial signal
(Fig.1H). This represented about 50-fold improvement in resist-
ance to photobleaching compared to the top-performing organic
dyes tested. As a result, S10C5H was chosen for subsequent experi-
ments. These QDs were 3.7±0.6nm in diameter and displayed the
chalcopyrite phase structure characteristic of bulk CuInSe2 (Fig.1I
and fig. S4).
QD encapsulation
After synthesis, a subset of S10C5H QDs was encapsulated in
poly(methyl methacrylate) (PMMA) microspheres using a sponta-
neous emulsion/solvent evaporation technique (Fig.2,AtoD).
Emulsion parameters such as surfactant concentration and homoge-
nization speed were refined to produce particles that contained
60% QDs by mass with an average size of 15.7±5.3 m (Fig.2E).
Encapsulated QDs displayed a similar but slightly red-shifted
emission peak due to Förster resonance energy transfer in closely
packed QDs (Fig.2F). Despite the about 40% loss of signal per mass
due to the presence of PMMA, these particles still exhibited bright
fluorescence (Fig.2G), which was stable for months in phosphate-
buffered saline (PBS) at 37°C (Fig.2H). QDs embedded in PMMA
also demonstrated consistent fluorescence intensity over a pH range
that would be physiologically relevant in the phagolysosome (fig. S5).
Microneedle geometry optimization
To determine the optimal microneedle geometry for this applica-
tion, finite element analysis was performed. Fifty shapes ranging
from a cone to a cylinder at a fixed height of 1500 m were mechan-
ically analyzed to ensure deep delivery of QDs into a permanent
(nonshedding) layer of skin (figs. S6 and S7). Important design crite-
ria were to provide ample resistance to mechanical failure while
also achieving a high deliverable volume, defined as the volume
near the dissolving microneedle tip. Finite element analysis identi-
fied that microneedles with diameters of 100 or 200 m were prone
to failure due to both bending and axial loading; therefore, all sub-
sequent studies used needles 300 m in diameter. Both the me-
chanical performance and the deliverable volume from the needles
were heavily dependent on (the proportion of microneedle height
that was cylindrical) (Fig.3 and figs. S6 and S7). Increasing gen-
erally contributed to higher deliverable drug volume, improved
buckling resistance, and decreased maximum stress and displace-
ment under axial loading while increasing maximum stress under
bending. From these simulations, a value of 0.5 was selected for
. The maximum von Mises stress for our chosen microneedle
geometry under bending and axial loading were 9.46 and 8.71 MPa,
respectively; the axial and bending displacement were found to be
negligible, and the critical load factor was 0.0146 (fig. S6). After
considering these factors along with deliverable volume, a micro-
needle with a 300-m-wide cylindrical base that was 750 m in height
with an upper cone 750 m in height was selected as a leading can-
didate for further study.
To verify in silico modeling data, we down-selected to three 3 ×
3 arrays of microneedles: A 1500-m cone (Fig.3,AandB), a
750/750-m cylinder/cone (Fig.3,CandD), and a 1250/250-m
cylinder/cone (Fig.3,EandF) were printed using two-photon
polymerization to create a master mold in photoresist. These shapes
were maintained when the photoresist masters were used to pro-
duce inverse polydimethylsiloxane (PDMS) molds and ultimately
positive dissolvable microneedles composed of poly(vinyl alcohol)
and sucrose (Fig.3G).
The force required to penetrate synthetic human skin or ex-
planted pig skin with nondegradable in silico–optimized needles
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(750/750-m cylinder/cone) was not significantly different from
conical needles (P=0.46 and P=0.07, respectively), which have a
lower deliverable volume, but substantially less force than the blunter
(1250/250-m cylinder/cone) needles was required (Fig.3H). De-
creasing the spacing of microneedles from 3.23 to 1.57mm (1cm
by 1cm and 0.5 cm by 0.5cm patch sizes, respectively) did not
affect the amount of force required for penetration (Fig.3I). As a
result, we confirmed our selection of microneedles with a height of
1500 m, diameter of 300 m, and of 0.5 as the best combination
of penetration depth, resistance to mechanical fracture, and max-
imum capacity for microparticle delivery.
Smartphone imaging system design and ex vivo
skin penetration
To enable the imaging of NIR QDs in a field setting, we designed an
inexpensive, smartphone-based imaging system. A 780-nm NIR
light-emitting diode (LED) was paired with an 800-nm short-pass
filter and aspheric condenser with diffuser to excite the QDs (Fig.4A).
For NIR fluorescence detection, a Nexus 5X smartphone (Google)
was stripped of its stock short-pass NIR filter and paired with an
850-nm long-pass color glass filter and an 850-nm long-pass dielectric
filter set in a poly(lactic acid) three-dimensional (3D)–printed phone
case (Fig.4B). Imaging a QD-loaded microneedle patch with and
Fig. 1. Platform schematic and fluorescent probe characterization. (A) Fluorescent microparticles are distributed through an array of dissolvable microneedles in a
distinct spatial pattern. (B) Microneedles are then applied to the skin for 2 to 5 min, resulting in dissolution of the microneedle matrix and retention of fluorescent micro-
particles. (C) A NIR LED and adapted smartphone are used to image patterns of fluorescent microparticles retained within the skin. By selectively embedding microparticles
within microneedles used to deliver a vaccine, the resulting pattern of fluorescence detected in the skin can be used as an on-patient record of an individual’s vaccination
history. (D) Rapid photobleaching of organic dyes covered with pigmented human skin under simulated solar light. (E) Emission profiles of QDs (solid lines) show a blue
shift with increased shelling time. Dashed line depicts absorption by the 5-hour shelling sample. Arrows indicate the relevant y axis for absorption (left) and emission
(right). a.u., arbitrary units. (F) PL QY as a function of shelling time under different excitation wavelengths. (G) Relative photoluminescence intensity comparison of QDs
with the commercial inorganic dyes IRDC2 and IRDC3 (blue bars) and corresponding emission peaks (empty purple circles and square). (H) Photostability of QDs covered
with pigmented human skin under simulated solar light. (I) Transmission electron microscopy (TEM) and high-resolution TEM (inset) showing the size and crystal structure
of ZnS:Al-coated CuInSe2 QDs. Scale bars, 20 and 5 nm, respectively. n = 3 for all graphs containing error bars. Error bars indicate SD.
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without the long-pass emission filters demonstrated the ability
to filter background light while retaining NIR QD signal transmis-
sion (Fig.4, Cand D). All imaging was performed under ambient
indoor lighting with no measures taken to reduce environmental
background.
We then tested the ability of microneedles to deliver fluorescent
microparticles into explanted tissue samples. A simple spring-loaded
applicator was used to administer microneedles into exvivo pig
skin and human cadaveric skin for a duration of 2 min, in accor-
dance with the optimal wear time identified by the global health orga-
nization PATH (19). Before application, microneedles appeared sharp
with optically dark tips where QD-loaded microparticles were embedd ed
(Fig.4,EandF). After application, microneedles were blunted as a
result of the partial dissolution of the tip (Fig.4,GandH). Although
many fluorescent microparticles remained within the body of the
needle, the transfer of QD-loaded particles from the 4 × 4 microneedle
array into both pig skin (Fig.4,ItoL) and pigmented human skin
(Fig.4,MtoP) was obvious when imaged using our NIR-adapted
smartphone camera, yet not apparent by naked eye, as intended.
Pattern imaging, manual analysis, and machine
learning–based pattern recognition
After confirming penetration and dissolution exvivo, an 8-day
study was performed to assess invivo delivery. Microneedle patches
containing QDs were administered to the rear flank of Wistar rats,
as shown in movie S1. Before administration, hair at the application
site was removed using an electric razor and depilatory cream.
Microneedles were applied using a spring-loaded applicator and
held in place for 2min to allow for partial dissolution. This study
revealed the importance of encapsulating QDs to ensure their deliv-
ery and prolonged residence in the body. Unencapsulated QDs
resulted in very little and inconsistent transfer into the skin, likely
due to their hydrophobicity (Fig.5A). Alternatively, encapsulated
QDs showed a much higher transfer of fluorescence into the skin
that resulted in a NIR signal that was 10-fold higher than unencap-
sulated QDs (Fig.5B). Over the days after administration, the signal
from unencapsulated QDs was no longer apparent after 24 hours,
whereas the signal from encapsulated QDs decreased on the first
2 days after application but then seemed to stabilize (Fig.5C).
Next, to test the longevity of QD-containing PMMA microparti-
cles invivo, we administered patches containing eight microneedles
in one of three distinct patterns—a circle on day 0, a cross on day 28,
and a rectangle on day 56—and performed longitudinal imaging over
9 months (Fig.5,DtoI). On the day of application, markings were
visible on the skin for 100% of needles (120 of 120) with very high
contrast. By the 24-week time point, 92% (110 of 120) remained
visible with somewhat lower contrast than on the day of application.
The number of marks per pattern at this time was 7.3±0.8, which
was largely independent of the spatial distribution of microneedles
at the time of administration (7.4±0.8, 7.2±0.8, and 7.4±0.8 for
the circle, cross, and rectangle, respectively) (Fig.5J). Fluorescent
marks exhibited an average signal that was 29-, 23-, and 29-fold
higher than background at 3, 6, and 9 months after administration,
respectively, when considering the aggregate of all patterns (Fig.5K).
There were no statistical differences between the signal at 3 and
6 months (P=0.53), 3 and 9 months (P=0.998), or 6 and 9 months
(P=0.50).
Although patterns were easy to manually identify, we aimed to
automate this process to improve its ease of implementation and
thus potential clinical impact. To eliminate the need for personnel
training and minimize opportunities for human error, we developed
a machine learning algorithm based on the AlexNet neural network
(20) to automatically classify each pattern (fig. S8). Using this neu-
ral network, we were able to correctly classify 100% of test image
patterns (210 images in total) that were collected biweekly for up to
30 weeks. In addition to correctly classifying all patterns, the prob-
ability of those classifications was also very high, with no trend
toward lower probabilities over time (Fig.5L). Of the 210 images
Fig. 2. Encapsulation and characterization of QDs in PMMA microspheres. Light microscopy images of PMMA microspheres loaded with (A) 0% (w/w) QDs, (B) 37.5%
(w/w) QDs, and (C) 60% (w/w) QDs. (D) SEM image of PMMA microparticles containing S10C5H QDs. (E) Histogram of volumetric particle distribution smoothed using an
11-frame moving window smoothing function for improved clarity, n = 104 particles analyzed. (F) Photoluminescence profiles of S10C5H QDs before and after PMMA
encapsulation showing a minimal shift in fluorescence emission wavelength. (G) Relative photoluminescence intensities (blue bars) using an 850-nm long-pass filter
(n = 2) and emission peaks (empty purple circles). (H) Maintenance of photoluminescence intensity in PBS at 37°C over the course of months (n = 3), with representative
NIR images inlaid at their respective time points; *P < 0.05 (one-way ANOVA with Tukey’s multiple comparisons). Dashed line indicates the camera saturation point. Error
bars indicate SD.
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analyzed, the lowest classifications probability for image was 98.4%
for the cross pattern in one rat on day 154. These data suggest that
changes in signal intensity occur soon after administration (within
3 months) but that this does not affect detection reliability. This is
also supported by qualitative images shown in Fig.6 that depict
dimmer signals between 0 and 12 weeks but no further substantial
losses between 12 and 24 weeks.
QD-loaded microparticle biocompatibility and codelivery
with polio vaccine
We then sought to test the invitro and invivo biocompatibility of
our QDs. In vitro evaluation of cytotoxicity confirmed that our
custom QDs composed of CuInSe2 cores and ZnS:Al shells were less
toxic to macrophages than commercially available PbS QDs with a
similar oleic acid surface treatment (Fig.7A). Whereas no dose-
dependent toxicity was observed in the range of S10C5H QD con-
centrations tested, we did observe a trend in PbS QD toxicity with a
significant drop in cell viability at 1000 g/ml (P<0.05).
Histological examination of the local tissue response revealed
tissue damage consistent with needle penetration 1 day after admin-
istration (Fig.7,BandC). A minimal foreign body reaction was
observed at 2 weeks (Fig.7,DandE) and 4 weeks (Fig.7,FandG).
At both later time points, there appeared to be a small number of
macrophages and foreign body giant cells at the site of administra-
tion. No fibrous encapsulation was observed over the time points
collected, supporting the biocompatibility of the microparticles. In
general, these results suggest that the PMMA-encapsulated S10C5H
QDs largely remain local and are well tolerated by the body. These
observations were in agreement with visual observation of live
animals over the days after microneedle administration, which showed
no obvious signs of irritation beyond the date of application.
To observe the compatibility of this approach with vaccine delivery,
we evaluated the immune response to three doses of microneedle-
delivered Salk inactivated poliovirus vaccine type 2 (IPV2) admin-
istered at 0, 1, and 2 months with or without QD-loaded microparticles.
IPV2 coadministered with microparticles induced total and neu-
tralizing IPV2 antibody titers that were not statistically different
(P=1.00 and P=0.91, respectively) from those achieved by IPV2
delivered by microneedles alone (Fig.7,HandI). Total and neutral-
izing titers induced by microneedles containing QD-loaded micro-
particles and IPV2 were also noninferior to three subcutaneous
injections of IPV2 (P=0.25 and P=0.32, respectively) despite the
use of a suboptimal formulation, suggesting a strong dose-sparing
effect because only 25±2% of the vaccine retained its D-antigenicity
during microneedle fabrication and a large fraction of IPV2 remained
undelivered within the incompletely dissolved microneedles. Despite
the substantially lower dose of antigen delivered in its immunity-
conferring conformation, the neutralizing antibodies achieved were
well above the threshold considered protective by the U.S. Centers
for Disease Control and Prevention (21).
DISCUSSION
To maximize the utility of this technology for vaccination campaigns,
we aimed to create a platform compatible with microneedle-delivered
vaccines that could reliably encode data on an individual for at least
Fig. 3. Microneedle modeling, fabrication, and evaluation. Optical images of microneedles and finite element analysis data of (A and B) a conical needle 1500 m in
height and 300 m at its base; (C and D) a microneedle 300 m at its base with a 750-m cone atop a 750-m cylinder; and (E and F) a microneedle 300 m at its base with
a 250-m cone atop a 1250-m cylinder. (G) SEM image of a dissolvable microneedle array based on the geometry shown in (C). (H) Ex vivo penetration force per needle
based on microneedle geometry; n = 3; ***P < 0.001 and ****P < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons). (I) Spacing-independent penetration force
requirements in pig skin ex vivo (Student’s t test). In (B), (D), and (F), 0 and 100 represent the worst and best values, respectively, for each parameter for between 0 and 1.
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5 years after administration. In addition, this system also needed to
be highly biocompatible, deliver a sufficient amount of dye after an
application time of 2min or less, and be detectable using a minimally
adapted smartphone. Given the limitations of organic dyes (photo-
bleaching and water solubility) and inorganic dyes [rare earth metal
toxicity (22), low fluorescence, and processing difficulty], QDs were
an attractive option as a potentially bright, photostable, and tunable
alternative. However, clinical implementation of QDs has been stymied
by the toxicity of their core elements, such as cadmium and lead (23).
To overcome these safety concerns—which are especially important
because these materials would be given to healthy children—we
chose to synthesize custom QDs composed of more well-tolerated
elements. We identified an optimal fluorescent emission range of
850 to 1100nm. Operating in this range mitigates the potential cul-
tural opposition to visible skin markings, reduces background from
ambient light, minimizes light absorption by tissue at wavelengths
below 850nm (24), and maximizes signal detection by avoiding the
poor sensitivity of inexpensive silicon-based detectors to light above
1100nm (25). Although the use of fluorophores at the higher end of
the NIR-II window (1000 to 1700 nm) would further improve tissue
transparency and reduce background (26), appropriate detectors for
these wavelengths (indium gallium arsenide) are typically considered
cost prohibitive (27), which could render this approach infeasible for
widespread implementation.
Fluorophore photobleaching under accelerated solar light expo-
sure enabled us to predict signal loss associated with sun exposure
over long periods of time. As expected, organic fluorophores de-
graded relatively quickly despite protection by heavily pigmented
skin, which absorbs a large fraction of ultraviolet (UV) and visible
light. These dyes also exhibited strong self-quenching in the dry state
and therefore had to be studied in a dispersed, hydrated state. This
self-quenching property is problematic for this microneedle-delivered
Fig. 4. Smartphone modifications and NIR marking detection in skin. (A) Photograph of disassembled LED used for NIR illumination at 780 nm combined with an
800-nm short-pass filter and aspheric condenser. (B) Photograph of disassembled NIR imaging smartphone consisting of a Google Nexus 5X smartphone with the internal
short-pass filter removed and replaced with two external 850-nm long-pass filters set in a 3D-printed phone case. Images of a 16-needle microneedle patch containing
PMMA-encapsulated QDs were collected with the adapted smartphone under ambient indoor lighting (C) without the 850-nm long-pass filters and (D) with the pair
of 850-nm long-pass filters under LED illumination from the same distance. Inset shows an image at a higher exposure. (E) Optical and (F) SEM images of fluorescent
microparticle-loaded microneedles before skin application. (G) Optical and (H) SEM images of microneedles after administration to explanted pig skin. Adapted smartphone
images of pig skin before microneedle application (I) without and (J) with 850-nm long-pass filters. Adapted smartphone images of pig skin after application (K) without
and (L) with 850-nm long-pass filters. Adapted smartphone images of pigmented human skin before microneedle application (M) without and (N) with the 850-nm long-
pass filters. Smartphone images of human skin after application (O) without and (P) with the 850-nm long-pass filters. Note: Scale bars in NIR-filtered images are approx-
imate with (J), (L), (N), and (P) taken at about the same distance. Components in (A) and (B) cropped for clarity.
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recording system because it limits the packing density of the dye
and thus its brightness. Alternatively, QDs performed very well
under light exposure and have a larger Stokes shift, which enables
them to avoid substantial reabsorption and therefore be used in a
densely packed format.
Although the accelerated photobleaching assay does not perfectly
mimic the real-world use case, it does provide evidence supporting
the longevity of QDs compared to organic dyes. Further, this exper-
imental setup may overestimate photobleaching because it assumes
that skin is in direct sunlight every hour of the day that the sun is out.
If administered at the thigh, where current vaccines are administered
to infants, this area may be covered by clothing and/or in an area that
is not exposed to direct sunlight for some portion of the daytime. In
addition, photobleaching may also be enhanced in this experiment
because sevenfold more intense light results in greater energy transfer
per time, potentially enabling QD degradation to proceed more quickly.
PMMA was used as a nondegrading, encapsulating material for
QDs to improve biocompatibility and enhance tissue permanence.
Fig. 5. In vivo imaging of NIR patterns in rodent skin. Administration site after the delivery of a 4 × 4 microneedle patch containing (A) unencapsulated QDs or
(B) PMMA-encapsulated QDs. (C) Short-term study of signal intensity after microneedle application of unencapsulated or PMMA-encapsulated QDs to rat skin, n = 4.
Images of (D) circle, (E) cross, and (F) rectangle patterns imaged 24 weeks after administration of PMMA-encapsulated QDs to rats. Log-scale color maps of the same
(G) circle, (H) cross, and (I) rectangle patterns shown in (D) and (F). (J) Number of markings detected 24 weeks after administration, n = 5. (K) Quantification of signal-to-
noise ratio for the circle pattern showing no changes between 12, 24, and 36 weeks, n = 15 (one-way ANOVA with Tukey’s multiple comparisons). (L) Graph showing
the average probability of the machine learning algorithm (all patterns correctly detected), n = 5. Grayscale images extracted from red channel of the adapted
smartphone-generated Red Green Blue (RGB) image.
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The large size of microparticles (15.7±5.3 m)
was also hypothesized to minimize clear-
ance because previous studies have shown
that larger particles are more resistant to
clearance by macrophages, the most rel-
evant cell type for clearing foreign mate-
rial (28). The flexibility of QD surface
coating also enabled us to make QDs
soluble in the same organic solvent as
PMMA, which helped markedly enhance
loading given that a traditional double
emulsion technique (water-in-oil-in-water)
cannot achieve 60% (w/w) loading.
We hypothesized that, unlike microneedle-
delivered vaccines, which must simply
break the skin’s water barrier to deliver
their payload effectively (29), these fluo-
rescent microparticles must be delivered
below the shed layers of skin to ensure their
long-term residence in tissue. Therefore,
we aimed to create needles 1500 m in
length. To successfully penetrate to this
depth, the microneedles must resist me-
chanical stress, deflection, and displace-
ment upon insertion into skin. To effectively
deliver PMMA-encapsulated QDs into
the skin, the microneedles must pierce
the skin without fracturing. The critical
load factor of 0.014557 was higher than
the values reported for carboxymethyl
cellulose microneedles using the same
simulation procedures (30). The final se-
lection of =0.5, height=1500 m, and
width=300 m avoids critically low me-
chanical robustness against all the major
mechanisms of mechanical failure. Using
a larger diameter and larger increased
the volume of material and thereby the
number of QD-loaded particles that are
available for transfer into the skin. The
drawback of this is the potential increase
in pain and higher penetration force re-
quirements. However, although pain per-
ception has been shown to increase with the
diameter of hypodermic needles (3133),
the amount of pain induced by our mi-
croneedle patch is likely less than for
Fig. 6. Longitudinal imaging of NIR markings in
rodent skin. Cropped, but otherwise raw, smartphone
images collected from a fixed distance show ing the
intradermal NIR signal from PMMA-encapsulated
QDs delive red via microneedle patches on rats 0,
12, and 24 weeks after administration. The text at
the bottom of each image indicates the image
collection settings ISO density and shutter speed
(SS) in seconds.
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traditional needles, as has been shown elsewhere (34). Further, a
previous study has shown that tip angle does not significantly affect
pain upon needle insertion (35), so the effect of (effective tip
angle) on pain perception was not considered. These needles are
slightly smaller in diameter than a 30-gauge needle and therefore
should minimize both pain and the force required for skin penetra-
tion (36). Last, the lateral microneedle spacing results in a patch,
and subsequently intradermal pattern, with a footprint of 0.25cm2.
This spacing was sufficiently large to allow each needle to act inde-
pendently and thereby allow force requirements to scale linearly
with needle number (37).
Over the past decade, smartphones have become ubiquitous in
many areas of the world, including the developing world despite
limited infrastructure (38). Because these phones offer on-board
processing power, camera applications, and inexpensive consumer-
grade camera modules, we chose to adapt an existing smartphone to
enable NIR imaging rather than build a completely new imaging
system. In addition, we believe that familiarity with the function of
these devices will lessen the learning curve for NIR imaging in a
field setting. Whereas a stock Nexus 5X camera is built with a short-
pass filter to prevent NIR light from affecting images, we wanted
the exact opposite—the elimination of light in the visible range
and passage of NIR light. In addition, we needed the new filters
to block reflected light from LED illumination. Therefore, after
stripping the stock short-pass filter, we added a pair of 850-nm
long-pass filters, which would block both environmental light and
LED illumination. A dielectric filter was used to impose a sharp
cutoff at 850nm and paired with an 850-nm color glass filter to
eliminate the passage of visible light entering the filter at very small
angles, which we observed to be problematic with the dielectric filter
alone (39). These optics were fit into a 3D-printed phone case
customized for SM1-threaded components (1.035″-40) to fit with
commercially available optical components.
Because both excitation and emission light can be absorbed by
the body, we needed to create a system where both stages were within
the optical imaging window. Although QDs are broadly excitable
and exhibit higher quantum yield when exited at lower wavelengths
(UV and visible light), high absorption from tissue components
such as hemoglobin, water, and melanin at these wavelengths greatly
attenuates the signal. Given previous studies examining the relative
absorption of these components, excitation at higher wavelengths
was deemed worth the trade-off. For example, our leading candidate,
S10C5H, exhibited a PL QY of 43.6±0.1% upon excitation with
a 405-nm laser and a PL QY of 31.3± 0.1% when excited with an
808-nm laser, but would be subjected to an increase in absorption from
hemoglobin and melanin on the order of one log unit each (40). We
chose to use a 780-nm LED rather than a laser because of the
reduced cost, safety concerns, and maintenance combined with
improved portability. Adapting a laser to this setup would have
required a diffusor or beam splitter to spread the light, safety mech-
anisms, and potentially some type of cooling apparatus. These com-
ponents would add cost, complexity, and size, which are undesirable
for distributed mobile use. Alternatively, an LED offered low cost,
inherently diffuse illumination, and safety/power advantages. The
only consideration necessary to use an LED was the considerably
wider emission profile (28-nm full width at half maximum); however,
this was solved using a simple 800-nm short-pass filter to ensure a
50-nm gap between excitation light and light collected by the adapted
smartphone.
Fig. 7. Biological response to PMMA-encapsulated QDs. (A) In vitro cytotoxicity
of commercially available PbS QDs compared to unencapsulated and PMMA-
encapsulated S10C5H QDs over 24 hours in a mouse macrophage cell line (Raw 264.7) .
n = 3, *P < 0.05, ***P < 0.001, and ****P < 0.0001 (two-way ANOVA with Tukey’s multiple
comparisons). Representative histological samples collected from rats receiving
microneedle-delivered PMMA particles containing S10C5H QDs (B and C) 1 day,
(D and E) 2 weeks, and (F and G) 4 weeks after administration stained with hema-
toxylin and eosin or Masson’s trichrome, respectively. Arrows indicate the location of
microparticles. (H) Total anti-poliovirus type 2 immunoglobulin G antibody titers and
(I) neutralizing poliovirus type 2 antibody titers showing no differences after three
doses of IPV2 delivered via subcutaneous injections or microneedles with or without
PMMA-encapsulated QDs, n = 5. Dashed line indicates the threshold above which
humans are considered immune.
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NIR images collected with the smartphone at various ISO and
shutter speed ratings were manually cropped to enable the compar-
ison of NIR signal intensity over time. Manual ISO and shutter
speeds were necessary to enable longitudinal comparison. The auto-
exposure setting was not well adapted for NIR imaging, as might be
expected, and would often select higher exposure settings to make
the field brighter to the point where the background was bright and
the signal was highly saturated. Selection of the correct ISO for a
pattern was critical for accurate analysis of both signal-to-noise
ratio (SNR) as well as machine learning classification. To enable
accurate SNR quantification, consistent imaging settings that pro-
duced nonsaturated pixels at all time points were selected. In the
future, custom autoexposure settings could be implemented to
automate this process.
The greatest reduction in user-identifiable signal occurred soon
after administration, suggesting that some microneedles had not
delivered QD-loaded microparticles to a sufficient depth—a phe-
nomenon possibly exacerbated by the high elasticity of rodent skin
(41). A portion of particles may have been deposited on the skin
rather than in the skin and thereafter cleared by external perturba-
tion. The QD signal stabilizes rather quickly and then presents a
fairly consistent SNR at 3, 6, and 9 months, indicating that micro-
particle clearance likely primarily occurs at early time points (less
than 3 months). This is further supported by the imaging of patterns
invivo at short time points, which showed considerable loss in the
days after application before stabilizing.
AlexNet was chosen for its classification accuracy, having been
originally trained on more than 1 million images and having demon-
strated the ability to classify images into 1000 categories (20). Despite
relatively little training data (30 images per pattern), AlexNet classi-
fied images with high accuracy. Overall, 80 images were tested for
the circular pattern, 70 for the cross pattern, and 60 for the rectan-
gular pattern originating from five distinct applications per pattern.
The modified transfer-based convolutional neural network using
AlexNet provided accurate detection confidence. Further, because
there was no trend toward lower machine learning classification
probability at 3, 6, and 9 months, it appears that the patterns are
stable after an acute period of signal loss, which is also supported by
short-term quantitative data and long-term qualitative data. On the
basis of optical images of microneedles before and after administration,
invivo NIR imaging, and projections for photobleaching from invitro
experiment, we estimate that <1 g of particles (<600ng of QDs) is
required to retain a detectable signal for 5 years.
Regarding accuracy, machine learning algorithms for image
classification match and typically exceed manual inspection (42, 43).
Whereas the human eye is ill equipped to distinguish low grayscale
value differences in images that are dim or have poor contrast
(for example, grayscale values of 1 versus 10), AlexNet’s ability to use
quantitative rather than qualitative information seems to make this
distinction trivial. This effect is readily apparent when the grayscale
photo values are represented as a rainbow heat map. AlexNet was
particularly valuable when markings were missing, dim, or imaged
at an unusual angle. Given the minimal training data used, it was
important that images were collected using settings that roughly
approximated the range of brightness/signal spread of images used
for training. More training data and/or consistent autoexposure would
mitigate this potential issue long term. Similarly, more patterns could
be incorporated into the model to expand the variety of data that can
be encoded. In the real-world use case, this automated classification
will eliminate classification subjectivity that could interfere with the
accuracy of this approach. Synthetic data could also be produced
using image augmentation to train the algorithm without the need
for actual patch applications. However, the accuracy of this method
could be lower if the variability in application exhibited in the syn-
thetic data did not replicate the features of real microneedle patch
applications in the test data. In our experience, training AlexNet
with 5000 synthetic images yielded a real-world data classification
accuracy of 92% for the circle and 97% for both the cross and the
rectangle. Future training data could also include a fourth classification
category for all images that do not closely resemble any of the pat-
terns. This would be essential for real-world use when the presence
of a pattern is uncertain.
For this encoding system to be clinically useful, microneedles
and NIR microparticles must be biocompatible and, if applicable,
maintain the utility of any codelivered vaccines. Histology showed
that the particles were well tolerated in the body, similar to largely
inert PMMA-based tattoo dyes (44), although some macrophages were
observed. The accumulation of macrophages observed around the
particles was less than other studies have shown for poly(lactic-co-
glycolic acid) microparticles (45), for example, which are present in
many U.S. Food and Drug Administration–approved drug delivery
systems (46). In addition, histological images demonstrated that
microneedle-delivered particles were delivered to a depth similar to
professional tattoos (47), which supports their potential for long-term
NIR detection.
In addition to the stand-alone value of an intradermal informa-
tion encoding and detection platform, this system may offer greater
advantages when codelivered with vaccines. By delivering both
agents in the same microneedle patch, there is the potential to realize
production cost advantages and eliminate the possibility of misuse
(such as applying the encoding patch without the vaccine). However,
to function in combination with microneedle vaccines currently under
development, PMMA-encapsulated NIR QDs must not interfere
with the robust immune response generated by intradermal antigen
delivery. To ensure sufficient QD delivery and vaccine stability after
fabrication (a process that includes multiple days under high vacuum),
the amount of sucrose in the microneedle formulation and appli-
cation time were both increased. Despite these modifications, a
decrease in vaccine stability (IPV2 D-to-C conversion) was still
observed and a considerable microneedle volume was not trans-
ferred to the skin during application, which would be expected
to reduce the magnitude of the immune response. Nevertheless,
microneedle-treated groups yielded total and neutralizing antibody
titers against IPV2 that were similar with theoretical dose-matched
subcutaneous injections and were above the threshold known to
confer protection in humans, regardless of the presence of encapsu-
lated QDs. This was observed despite substantial losses during
formulation and administration and is likely due to the dose-sparing
effects of intradermal delivery (16,17). Although the addition of
polymer could have had an adjuvant effect, this was not observed,
likely because of the well-tolerated, largely inert nature of our
PMMA microparticles, as demonstrated in our histological data. These
results support the clinical potential for using these microneedles
to coadminister an invisible marking agent and a vaccine in one
application.
This study provides support for long-lived, reliable pattern de-
tection using QDs; however, like many preclinical studies, it is lim-
ited in duration and relies on small animal models. We present data
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characterizing signal expression over 9 months in animals and invitro
accelerated solar light photobleaching, which suggest that detectable
markings would persist to the target time point of 5 years. On the
basis of histology and longitudinal imaging studies, we would not
anticipate a substantial decrease in signal within 5 years after appli-
cation; however, we did not explicitly demonstrate this because of
time constraints, which exceed the typical life span of our animal
model.
In summary, we have demonstrated proof of concept for a
platform capable of invisibly recording data in the skin. To establish
this system, we synthesized, encapsulated, and delivered cadmium-free
and lead-free photostable QDs into the skin using a custom micronee-
dle array, adapted a smartphone to create an inexpensive NIR imaging
system, and demonstrated the ability to accurately detect patterns
invivo for a period of 9 months using a semiautomated machine
learning algorithm. Transferring this technology to the clinic will
require several additional steps including preclinical safety and toxi-
cology studies, manufacturing scale-up, and first-in-human studies.
Additional invivo small animal studies will aid in the robust char-
acterization of local and systemic responses to QD-loaded PMMA
microparticles and microneedle patches to ensure quality, safety,
and reliability. Formative studies in which users test the devices,
including the packaging and labeling, will need to be conducted to
improve components for commercialization and to manufacture
devices for testing in human studies. Ultimately, we believe that this
invisible, “on-body” technology opens up new avenues for decen-
tralized data storage and biosensing applications that could influ-
ence the way medical care is provided, especially in the developing
world.
MATERIALS AND METHODS
Study design
These studies were designed to evaluate the suitability of intradermal
NIR fluorescent microparticles as a reliable, long-term detection
method. To evaluate this technology under typical field conditions,
we used an adapted smartphone in ambient light to determine suc-
cess criteria, except in circumstances where spectral and/or highly
quantitative data were necessary to characterize our materials. In
vivo studies were generally preferred to assess the performance of
our delivery and detection systems and animals were randomly
assigned to different experimental groups at the onset of each study.
The use of machine learning for detection eliminated the need for
blinding. In vitro studies were performed with three replicates and
invivo studies with five replicates, unless otherwise noted. Individual
subject-level data are reported in data file S1.
QD synthesis
Stoichiometric CuInSe2 cores were synthesized by mixing 1.5 mmol
of copper(I) iodide and 1.5 mmol of indium(III) acetate in 1.5ml of
1-dodecanethiol (DDT) and 45ml of 1-octadecene (ODE) based on
a modified procedure (48). The reaction mixture was then degassed
under vacuum for 20 min, purged with nitrogen for 20 min, and
degassed for an additional 45min at 120°C. Next, 1.5ml of oleic
acid was added into the mixture, and the solution was degassed for
20min. After another 20-min nitrogen purge, the solution was
heated to 175°C before the injection of selenium stock solution. A
selenium stock solution was first prepared by mixing 3 mmol of
selenium powder and 3ml of oleylamine (OLA) in 3 ml of DDT
(49). The selenium solution was degassed under vacuum for at least
30min at 60°C before injected into the reaction mixture. The reaction
mixture was heated to 200°C and maintained at this temperature
for 30min under the protection of nitrogen.
ZnS:Al shells were formed around CuInSe2 cores through the
dropwise addition of a stock solution containing zinc and aluminum.
The zinc precursor of the stock solution was prepared by mixing
30 mmol of zinc acetate in 30ml of OLA and 30ml of ODE, degas-
sing for 20 min, and purging with nitrogen for 20min. Next, the
solution was heated to 120°C and degassed under vacuum. The
Al precursor mixture consisting of 9 mmol of Al(IPA)3, 5.4ml of
DDT, and 36ml of ODE was degassed for 20min and purged with
nitrogen for 20min. The vessel was then sealed and sonicated for
1hour at 60°C (50). The aluminum stock solution was then added
to the zinc stock solution using a glass syringe and long needle.
After mixing the zinc and aluminum precursors, the resulting
shell stock solution was added dropwise to the reaction mixture of
CuInSe2 cores at 0.1 ml/min using a pump. At the same time, 15ml
of DDT was added at a rate of 0.5 ml/min to thermally trigger the
release of sulfur (51). The reaction was allowed to proceed for vary-
ing amounts of time, depending on the formulation. Afterward, the
reaction solution was cooled to room temperature and precipitated
twice into acetone, once into a 50:50 solution of acetone and meth-
anol, and twice more into methanol. Between each round of pre-
cipitation, QDs were resuspended in a minimal amount of toluene
and oleic acid that was added dropwise until the solution turned
transparent. After the final precipitation step, QDs were dispersed
in toluene.
Photobleaching analysis
Organic dyes required different imaging conditions than inorganic
dyes and QDs because of their small Stokes shift and therefore high
propensity to self-quench in the dry state. Organic fluorophores
were dissolved in water at a concentration of 10 g/ml, which was
found to be within the linear absorbance range for all dyes. Fifty
microliters of each dye was added to a black-walled 384-well plate,
covered with a 1-mm-thick quartz slide, and sealed with parafilm at
the edges to prevent evaporation during light exposure. The plate
was then read using a Tecan Infinite M200 Spectrophotometer to
determine the fluorescence intensities of Alexa Fluor 790 [784/814-nm
excitation/emission (ex/em)], DyLight 800 (760/810-nm ex/em),
IRDye 800CW (768/798-nm ex/em), IR-820 (710/820-nm ex/em),
Sulfo-Cyanine7 (750/773-nm ex/em), VivoTag 800 (785/815-nm ex/
em), and indocyanine green (805/835-nm ex/em).
Samples were then placed under a 300W PV Cell Testing Solar
Simulator Model 16S-300-002 (Solar Light). The ozone-free short-
arc xenon lamp was used with an air mass 1.5 filter and focusing
lens to expose samples to conditions mimicking the spectral distri-
bution and power density of sunlight at sevenfold the intensity of
the sun (695 mW/cm2). A 2-mm-thick piece of pigmented human
cadaver skin from donors age 21 to 68 years who self-identified as
African American was overlaid onto the sample to recapitulate
light absorption by tissue above an intradermal fluorophore. Skin
was kept hydrated by a continuous flow of water over the surface and
chilled from below using a CP-200HT-TT Peltier-Thermoelectric
Cold Plate Cooler (TE Technology) to prevent damage to the skin.
Samples were periodically removed from the solar simulator and
imaged using the spectrophotometer. This experiment was repeated
three times with one replicate each.
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Inorganic dyes and QDs were capable of being imaged in their
dry state, so a modified protocol was used. Holes were punched in a
black silicone sheet using a 2-mm stainless steel biopsy punch to create
space for dyes. The silicone sheet was then treated with air plasma
for 1min on high power at 500 mtorr. The sheet was then placed on
a 2.54cm by 2.54cm quartz slide, which allowed it to adhere in a
water-tight manner. Suspensions of Infrared Down-Conversion 2
(IRDC2) and IRDC3 at 10 mg/ml in water were deposited 2 l at a
time and allowed to dry until a total of 10 l had been deposited. The
same steps was performed for ZnS:Al-coated CuInSe2 QD solutions
at 10 mg/ml in toluene. Double-sided tape and parafilm were then
used to seal a glass slide to the other side of the black silicone. The
sample was then placed quartz slide-up beneath a piece of pigmented
cadaveric skin in a metal block under continuous hydration on a
cold plate cooler and exposed to light at sevenfold solar intensity. At
predetermined time points, samples were removed from light and
imaged using a custom NIR-imaging platform consisting of a 500-mW
laser emitting at 808nm and thermoelectric-cooled mount powered
by a LDC210C Laser Diode Controller and TED200C Temperature
Controller, a 15× achromatic Galilean beam expander, protected silver
mirror, 850-nm color glass long-pass emission filter, and a high-
sensitivity USB 3.0 complementary metal-oxide semiconductor camera
(DCC3240N) affixed to an optical breadboard (Thor Labs) inside a
dark work enclosure (U.S. Laser). Images were collected at different
exposure lengths for different dyes ranging from 10 to 2000 ms. The
longest exposure length that did not saturate the 10-bit pixel depth
was used for each dye at all time points. Owing to the length of
this experiment, all samples (n=3) were run simultaneously. For
all photobleaching experiments, skin was replaced every 2 to 4 days
to reduce the effects of melanin degradation. Intensity data were
quantified using ImageJ (National Institutes of Health) and normalized
by selecting a region of interest (ROI) around a well and comparing
the intensity above background in that image to the intensity above
background of the corresponding ROI at the beginning of the
experiment (52). All photobleaching data are reported as simulated
days of exposure based on a 12-hour light/dark cycle.
QD encapsulation
QD-loaded PMMA microparticles were formed using an oil-in-
water emulsion/solvent evaporation technique. Briefly, 150mg of
QDs and 100mg of PMMA were dissolved in 2ml of dichloromethane.
This solution was then added to 50ml of 1% poly(vinyl alcohol) (PVA)
[88% hydrolyzed, Mw (weight-average molecular weight), 31,000] solu-
tion in water. The resulting solution was emulsified at 5000 revolutions
per minute (RPM) for 1min using a T 18 digital ULTRA-TURRAX
homogenizer (IKA Works) and subsequently stirred at 250 RPM for
3 hours to allow solvent to evaporate. Particles were the centrifuged at
1000 relative centrifugal force (RCF) after which the supernatant was
removed. Particles were then washed four times by adding deionized
water, centrifugation at 1000 RCF, and supernatant removal. The re-
sulting particles were resuspended in deionized water and measured
using a Multisizer 3 (Beckman Coulter) with a 100-m aperture. Data
were smoothed using an 11-frame moving window and plotted as
a histogram with 300 equal-sized bins ranging from 2.1 to 59.9 m.
QD-loaded microparticles were then imaged using optical micros-
cop y and scan ning elect ron microscopy (SEM) to observe their shape.
Optical imaging was performed using an Olympus MX40 inspection
microscope with a ToupCam industrial digital camera (ToupTek
Photonics). In preparation for SEM, samples were deposited on
double-sided carbon tape and coated with a thin layer of Au/Pd using
a Hummer 6.2 Sputtering System (Anatech) to prevent charging.
Imaging was then performed using a JEOL JSM-5600LV scanning
electron microscope with an acceleration voltage of 5 kV.
The pH stability of S10C5H QDs in PMMA was determined by
casting a solution onto the bottom of a plate to prevent agitation
upon addition of the buffer. QDs (150 mg) and 100mg of PMMA
were added to 2ml of dichloromethane and sonicated for 5 min.
Using a glass pipette, about 5 l of the QD solution was added to the
bottom of a 96-well black-walled glass-bottom plate and dried over-
night. PBS (200 l) was then added to each well, and the samples
were incubated at 37°C for 24 hours. At this stage, the initial intensity
of the QD-PMMA samples were analyzed using a custom NIR-imagin g
platform as described above. To investigate the effect of pH on the
intensity of the QDs, the PBS was replaced by a buffer solution at
pH 4, 5, 6, 7.4 (PBS), or 10. The plate was then sealed to minimize
solvent loss and incubated at 37°C. After 1, 4, and 22 hours, samples
were removed from the incubator, and QD intensity was measured
using the custom NIR-imaging system with a 40-ms collection time.
Intensity data were quantified in ImageJ and normalized by selecting
an ROI around a well and comparing the intensity above background
in that image to the intensity above background of the corresponding
ROI at the beginning of the experiment.
Dissolvable microneedle fabrication and characterization
Water-soluble microneedle patches containing QD-loaded micro-
particles at the needle tips were fabricated using a solvent casting
process. First, microparticles were resuspended in water, pipetted
onto the top of the PDMS mold (4 l per microneedle), and centri-
fuged at 3234 RCF for 5min. Excess solution was cleared from the
top of the mold, and the solution was allowed to dry leaving behind
particles in the tips of the mold. This process was repeated when
loading was lower than desired or unevenly distributed. About 300 l
of a 17% (w/v) sucrose and 17% (w/v) PVA solution in water was
then dispensed on top of the mold and centrifuged at 3234 RCF for
5min. Molds were then left at room temperature overnight in a
laminar flow hood as an initial drying stage. Laser-cut acrylic discs
were then affixed to double-sided tape and attached to the back
side of the solidified microneedle patch. The patch was then carefully
removed from the mold and stored for an additional 72 hours under
vacuum desiccation.
Smartphone modifications
A smartphone camera was adapted with commercially available
optical components purchased from Thorlabs to enhance NIR QD
detection in the NIR. To enable NIR detection, the stock short-pass
IR filter was removed from a Google Nexus 5X smartphone. A smart-
phone case was then designed and 3D-printed to interface tightly
with optical components having SM1 threading directly in front of the
rear-facing camera. An 850-nm long-pass dielectric filter (FEL0850)
and 850-nm long-pass color glass filter (FGL850) were placed in
parallel in a lens tube attached to the smartphone case and held in
place with a retaining ring. For QD illumination, a 780-nm, 200-mW
mounted LED (M780L3) powered by a T-Cube LED Driver (LEDD1B)
and 15-V, 2.4-A power supply (KPS101) was used. To augment the
shape and spectrum of emission, an 800-nm dielectric short-pass filter
(FEL0800) and aspheric condenser lens with diffuser (ACL2520U-
DG6-B) were used in an adjustable lens tube. All imaging was per-
formed using the Camera FV-5 Lite app (FGAE Studios).
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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
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Biocompatibility of encapsulated QDs
Microneedles containing encapsulated QDs were administered to
Wistar rats (Charles River Laboratories) 8 to 12 weeks of age weighing
about 250 g to assess invivo biocompatibility. Rats were anesthetized
via continuous inhalation of 2.5% isoflurane throughout the admin-
istration and imaging process. Hair removal was performed before
microneedle administration by shaving the rear flank with an electric
razor and applying depilatory cream for about 2min. The area was
then rinsed to remove excess hair, sterilized using an ethanol swab,
and allowed to dry. Microneedles were applied using an MPatch
Mini spring-loaded applicator (Micropoint Technologies) for 2min.
Rats were returned to their cages and housed until terminal time
points at 1 day, 2 weeks, and 4 weeks (n=4 per time point). At the
time of sacrifice, rats were euthanized via CO2 asphyxiation. Skin was
explanted and fixed in formalin-free tissue fixative (Sigma-Aldrich)
for 24 to 72 hours. The relevant portion of the tissue was identified
using the adapted smartphone, transferred to 70% ethanol, and em-
bedded in paraffin wax. Samples were sectioned and stained with
hematoxylin and eosin or Masson’s trichrome. Interpretation of the
foreign body reaction was performed under the guidance of an ex-
perienced veterinary pathologist.
Longitudinal in vivo imaging of NIR patterns
Two longitudinal imaging experiments were performed to assess
the short-term loss of signal after administration and long-term res-
idence of microneedle-delivered particles. In both cases, QDs were
administered to rats using dissolvable microneedle patches as described
above and imaged periodically to observe signal intensity over time.
At each time point, rats were anesthetized under continuous inha-
lation of 2.5% isoflurane and imaged with the 780-nm LED and
NIR-adapted smartphone, maintaining a consistent imaging distance.
In the first experiment, microneedles containing PMMA-encaps ulat ed
and free (unencapsulated) ZnS:Al-coated CuInSe2 QDs were com-
pared to evaluate the potential benefits of encapsulation on initial signal
intensity and signal retention. Encapsulated QDs were embedded in
dissolvable microneedles using the protocol detailed above. Un-
encapsulated QDs were not readily dispersible in water, so the loading
procedure was slightly modified. Before loading, PDMS molds were
treated with air plasma using a Harrick Plasma Cleaner PDC-091-HP
on high power at 500 mtorr for 1min. QDs in toluene (4 l per
microneedle) were then deposited into the microneedle molds and
centrifuged at 3234 RCF for 5min. Toluene was then allowed to
dry. A solution of sucrose and PVA was then applied to the molds and
centrifuged into the needles and allowed to dry using the fabrication
steps outlined above.
After applying 16-microneedle patches, the highest nonsaturating
imaging settings were identified and used at all subsequent time
points for that group. For the unencapsulated group, these settings
were ISO 100 with a shutter speed of
1
30 s. For the encapsulated
group, these settings were ISO 100 with a shutter speed of
1
30 or
1
200 s. For each animal and time point, about 20 images were col-
lected with slightly different aim of the LED, which was controlled
independently from the camera that remained in a fixed location.
An ImageJ macro was then used to split images into their three con-
stituent red, green, and blue channels, save the red channel in gray-
scale format, and identify the images with the maximum intensity.
This image with the best aim was then used for subsequent quanti-
fication of signal-above background. Briefly, 80-pixel circular ROIs
were applied to each of the 16 dots in the 4 × 4 array to quantify the
average signal. The average grayscale intensity of the remaining pixels
was subtracted to generate longitudinally comparable values for signal
above background.
On the basis of this short-term study, a second long-term study
was performed using encapsulated QDs in eight-needle microneedle
patterns (circle, cross, or rectangle) for image detection and machine
learning analysis. Arrays were administered at 0 (circle), 4 (cross),
and 8 (rectangle) weeks to mimic a common vaccination schedule
in the developing world. A separate group was started in parallel to
generate an image dataset for training the machine learning algo-
rithm. Images were collected every 2 weeks at a variety of ISO and
shutter speeds, processed, and used for both SNR analysis and
machine learning classification.
Microneedle-based vaccine delivery
The solution used to cast microneedles was altered in favor of addi-
tional sucrose (2:1 w/w sucrose:PVA) to potentially improve vaccine
stability during processing. A solution of 34% (w/v) sucrose and
17% (w/v) PVA solution containing 3.2 DU of Salk IPV2 (MEF-1
strain; Statens Serum Institut) was centrifuged into PDMS micro-
needle molds using the procedures detailed above. After processing,
some microneedle patches were collected, resuspended in assay buffer,
and evaluated using a D-antigen–specific monoclonal sandwich
enzyme-linked immunosorbent assay (ELISA) for IPV2 to determine
the amount of the vaccine still in its immunity- conferring state. A
monoclonal antibody against IPV2 poliovirus (HYB 294-06-02,
Thermo Fisher Scientific) was used as both the capture and detec-
tion antibody, which was made possible because of the multiple
identical binding sites on IPV. To avoid species cross-reactivity, a
Lightning-Link Horseradish Peroxidase Kit (Novus Biologicals) was
used to prebind the detection epitope. Briefly, 100 l of antibody
diluted 1:1500in carbonate buffer (pH 9.6) was added to each well
of a Nunc Maxisorp LockWell 96-well plate and incubated over-
night at 4°C on an orbital shaker. Plates were then washed with PBS
containing 0.05% Tween 20 three times and incubated in 300 l of
blocking buffer containing the wash buffer and 5% (w/v) nonfat
milk for 1hour at 37°C. Plates were again washed and then loaded
with 50 l of samples. After 2hours of incubation at 37°C, plates
were washed five times and the Lightning Link–modified antibody
was added at 1:833in PBS. After 1-hour incubation, plates were
washed another five times and then 100 l of a SIGMAFAST OPD
substrate (Sigma-Aldrich) was added after resuspension according
to the manufacturer’s protocol. After a color change, 150 l of 1 M
sulfuric acid was added to each well to stop the reaction, and absor-
bance values were read at 490nm with a background reference of
630nm using a spectrophotometer.
Microneedle patches not used for ELISA were applied to rats for
a duration of 5min using the protocol described above. After appli-
cation, the portion of the microneedles remaining on the patch was
collected, resuspended in assay buffer, and measured via the IPV2
D-antigen ELISA. A control group of rats received a dose-matched
subcutaneous injection in the rear flank. These procedures were
repeated 4 and 8 weeks later to provide rats with second and third
doses. Rats were bled vial tail vein every 2 weeks after administra-
tion to collect serum for immunological analysis. Blood was collected
in BD SST tubes (product no. 365967, Becton Dickinson) and used
according to the manufacturer’s protocol to obtain serum, which
was subsequently stored at −20°C until use. A total anti-IPV2 anti-
body ELISA was performed in house using an indirect ELISA, as
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McHugh et al., Sci. Transl. Med. 11, eaay7162 (2019) 18 December 2019
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
14 of 15
previously described (53). IPV2 neutralizing antibody titers were
determined by the U.S. Centers for Disease Control and Prevention
using a protocol previously published (21).
Statistical analysis
Statistics were performed in GraphPad Prism using Student’s t test
for pairwise comparisons and one-way analysis of variance (ANOVA)
with Tukey’s multiple comparisons test for comparing multiple
groups at a significance level of =0.05. In vitro toxicity experi-
ments evaluating both QD type and concentration were analyzed
using two-way ANOVA at a significance level of =0.05. All
invitro experiments were performed in experimental triplicate
unless otherwise noted. All invivo experiments were performed with
five experimental replicates unless otherwise noted. Data are reported
in the text as means±SD.
SUPPLEMENTARY MATERIALS
stm.sciencemag.org/cgi/content/full/11/523/eaay7162/DC1
Materials and Methods
Fig. S1. Optical properties of organic dyes.
Fig. S2. Evolution of fluorescence emission properties with shelling time.
Fig. S3. Fluorescence lifetime characterization of the S10C QD series.
Fig. S4. Composition and physical properties of S10C5H QDs.
Fig. S5. pH stability of PMMA-encapsulated QDs.
Fig. S6. Finite element analysis of mechanical forces on microneedles.
Fig. S7. Optimization of microneedle geometry using finite element analysis.
Fig. S8. Machine learning training and validation.
Table S1. Spectral characterization of custom QD formulations.
Table S2. Multiexponential fitting parameters for photoluminescence decay curves.
Movie S1. Intradermal administration and imaging of encapsulated QDs.
Data file S1. Individual subject-level data.
References (5462)
View/request a protocol for this paper from Bio-protocol.
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62. Computer code associated with Biocompatible near-infrared quantum dots delivered to
the skin by microneedle patches to record vaccination; doi.org/10.5281/zenodo.3571386.
Acknowledgments: We acknowledge W. H. Gates, D. Hartman, S. Hershenson, S. Kern,
B. Nikolic, K. Owen, L. Shackelton, C. Karp, and D. Robinson for their guidance; R. T. Bronson for
pathology expertise; and the MIT Department of Comparative Medicine for advice. We thank
W. Weldon and his laboratory at the U.S. Centers for Disease Control and Prevention for
performing neutralizing antibody titer studies, the Koch Institute Swanson Biotechnology
Center for technical support, specifically the Hope Babette Tang (1983) Histology Facility and
the Peterson (1957) Nanotechnology Materials Core Facility as well as the Harvard University
Center for Nanoscale Systems, and W.M. Keck Microscopy Facility at the Whitehead Institute.
Funding: This work was funded by the Bill & Melinda Gates Foundation grant OPP 1150646.
Fellowship support for K.J.M. was provided by an NIH Ruth L. Kirschstein National Research
Service Award (F32EB022416). L.J. thanks the Youth Innovation Promotion Association CAS
(2018042), National Natural Science Foundation of China (81671755), and China Scholarship
Council (201604910444) for financial support. This work was supported in part by the Koch
Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. This work was
performed in part at the Harvard University Center for Nanoscale Systems (CNS), a member
of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported
by the National Science Foundation under NSF award no. 1541959. Author contributions:
K.J.M., R.L., A.J., L.W., and P.A.W. devised the concept. K.J.M., L.J., R.L., and A.J. designed the
experiments and wrote the manuscript. L.J. designed and synthesized QDs. L.J., H.S.N.J., C.F.P.,
and J.Y. performed optical characterization and analysis. M.G.B. and M.G. oversaw QD synthesis
and analysis. L.J., H.S.N.J., and W.T. performed QD encapsulation. M.S. performed the
computational modeling simulations. K.J.M., S.Y.S., and M.C. designed and fabricated
microneedles. K.J.M., S.Y.S., C.F.P., F.L., M.P., N.P., and A.K. designed the smartphone optics.
K.J.M., L.J., H.S.N.J., S.Y.S., S.Y.T., T.G., J.C., J.L.S., and M.T. performed the in vitro experiments.
K.J.M., S.Y.S., and M.C. performed the ex vivo studies. K.J.M., S.Y.S., M.C., S.R., and S.T. performed
the in vivo experiments and corresponding analysis. M.S. and D.V. developed the machine
learning algorithm. Competing interests: A patent application entitled “Microneedle tattoo
patches and use thereof” describing the approach presented here was filed by K.J.M., L.J., S.Y.S.,
H.S.N.J., A.J., and R.L. (US 62/558,172). R.L. discloses potential competing interests in the below
link: www.dropbox.com/s/yc3xqb5s8s94v7x/Rev%20Langer%20COI.pdf?dl=0.
Data and materials availability: All data associated with this study are present in the paper or
the Supplementary Materials. Computer code archive is publicly accessible: doi.org/10.5281/
zenodo.3571386.
Submitted 20 July 2019
Accepted 27 November 2019
Published 18 December 2019
10.1126/scitranslmed.aay7162
Citation: K. J. McHugh, L. Jing, S. Y. Severt, M. Cruz, M. Sarmadi, H. S. N. Jayawardena, C. F. Perkinson,
F. Larusson, S. Rose, S. Tomasic, T. Graf, S. Y. Tzeng, J. L. Sugarman, D. Vlasic, M. Peters, N. Peters on,
L. Wood, W. Tang, J. Yeom, J. Collins, P. A. Welkhoff, A. Karchin, M. Tse, M. Gao, M. G. Bawendi,
R. Langer, A. Jaklenec, Biocompatible near-infrared quantum dots delivered to the skin by
microneedle patches record vaccination. Sci. Transl. Med. 11, eaay7162 (2019).
at Rice Univ on December 18, 2019http://stm.sciencemag.org/Downloaded from
record vaccination
Biocompatible near-infrared quantum dots delivered to the skin by microneedle patches
Jaklenec
Joe Collins, Philip A. Welkhoff, Ari Karchin, Megan Tse, Mingyuan Gao, Moungi G. Bawendi, Robert Langer and Ana
Tzeng, James L. Sugarman, Daniel Vlasic, Matthew Peters, Nels Peterson, Lowell Wood, Wen Tang, Jihyeon Yeom,
Jayawardena, Collin F. Perkinson, Fridrik Larusson, Sviatlana Rose, Stephanie Tomasic, Tyler Graf, Stephany Y.
Kevin J. McHugh, Lihong Jing, Sean Y. Severt, Mache Cruz, Morteza Sarmadi, Hapuarachchige Surangi N.
DOI: 10.1126/scitranslmed.aay7162
, eaay7162.11Sci Transl Med
on-person vaccination recordkeeping.
identifiable using semiautomated machine learning. These results demonstrate proof of concept for intradermal
production. Discrete microneedle-delivered microparticle patterns in porcine and pigmented human skin were
intradermal delivery of microparticles in rats, and codelivery of inactivated poliovirus led to protective antibody
particles in the skin could serve as an on-person vaccination record. Patterns were detected 9 months after
invisible to the eye but can be imaged using modified smartphones. By codelivering a vaccine, the pattern of
microneedles that deliver patterns of near-infrared light-emitting microparticles to the skin. Particle patterns are
developed dissolvableet al.makes it challenging to track vaccine coverage across the world. McHugh
Vaccines prevent disease and save lives; however, lack of standardized immunization recordkeeping
On the record
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Supplementary Materials for
Biocompatible near-infrared quantum dots delivered to the skin by microneedle
patches record vaccination
Kevin J. McHugh, Lihong Jing, Sean Y. Severt, Mache Cruz, Morteza Sarmadi, Hapuarachchige Surangi N. Jayawardena,
Collin F. Perkinson, Fridrik Larusson, Sviatlana Rose, Stephanie Tomasic, Tyler Graf, Stephany Y. Tzeng,
James L. Sugarman, Daniel Vlasic, Matthew Peters, Nels Peterson, Lowell Wood, Wen Tang, Jihyeon Yeom, Joe Collins,
Philip A. Welkhoff, Ari Karchin, Megan Tse, Mingyuan Gao, Moungi G. Bawendi, Robert Langer*, Ana Jaklenec*
*Corresponding author. Email: rlanger@mit.edu (R.L.); jaklenec@mit.edu (A.J.)
Published 18 December 2019, Sci. Transl. Med. 11, eaay7162 (2019)
DOI: 10.1126/scitranslmed.aay7162
The PDF file includes:
Materials and Methods
Fig. S1. Optical properties of organic dyes.
Fig. S2. Evolution of fluorescence emission properties with shelling time.
Fig. S3. Fluorescence lifetime characterization of the S10C QD series.
Fig. S4. Composition and physical properties of S10C5H QDs.
Fig. S5. pH stability of PMMA-encapsulated QDs.
Fig. S6. Finite element analysis of mechanical forces on microneedles.
Fig. S7. Optimization of microneedle geometry using finite element analysis.
Fig. S8. Machine learning training and validation.
Table S1. Spectral characterization of custom QD formulations.
Table S2. Multiexponential fitting parameters for photoluminescence decay curves.
Legend for movie S1
References (5462)
Other Supplementary Material for this manuscript includes the following:
(available at stm.sciencemag.org/cgi/content/full/11/523/eaay7162/DC1)
Movie S1 (.mp4 format). Intradermal administration and imaging of encapsulated QDs.
Data file S1 (Microsoft Excel format). Individual subject-level data.
Materials and Methods
Materials and reagents
All chemicals used to synthesize and encapsulate quantum dots (QDs) and form microneedles
were purchased from Sigma-Aldrich with the exception of copper (I) iodide, indium (III) acetate,
oleic acid purchased from Alfa Aesar, poly(vinyl alcohol) (PVA) purchased form Polysciences,
and SYLGARD 184 silicone elastomer (polydimethylsiloxane) purchased from Dow Chemical
Company. Alexa Fluor 790 succinimidyl ester and DyLight 800 NHS ester were purchased from
Thermo Fisher Scientific. IRDye 800CW was purchased from LI-COR Biosciences. IR-820 was
purchased from Sigma-Aldrich. Sulfo-Cyanine7 NHS ester was purchased from Lumiprobe.
VivoTag 800 was purchased from PerkinElmer. Indocyanine green was purchased from MP
Biomedicals. IRDC2 and IRDC3 were purchased from LDP LLC.
Fluorophore characterization
Steady-state photoluminescence (PL) spectra were measured using a SpectraPro-300i single-
grating spectrometer (Princeton Instruments) in conjunction with a DET10N single-channel
indium gallium arsenide (InGaAs) detector (Thorlabs). All steady-state PL measurements were
conducted using 532 nm laser excitation, and emission was collected using a pair of gold-coated
off-axis parabolic mirrors. The photoluminescence quantum yield (PL QY) of the QDs was
obtained using an RTC-060-SF integrating sphere (Labsphere). The PL QY was measured using
405 nm laser excitation. QDs chosen for further study were also evaluated under 808 nm laser
excitation. Color glass long-pass filters were used to block the excitation beam. The light output
from the integrating sphere was detected using a calibrating 818-IR germanium detector
(Newport) through a SR830 lock-in amplifier (Stanford Research Systems). The sample was
placed in a polytetrafluoroethylene-capped quartz cuvette, and a solvent blank was used to
ensure that the environment inside the sphere was as uniform as possible. The measured
photocurrent was adjusted to account for the external quantum efficiency of the detector when
calculating the PL QY. The measured PL QY was then corrected to account for leakage of the
excitation light and the transmittance of the filter. Lastly, time-resolved PL decay was measured
using an LDH-P-FA-530B spectrometer (PicoQuant) equipped with a picosecond pulsed diode
laser (pulse width: 100 ps) as a single wavelength excitation source (532 nm) for time-correlated
single-photon counting measurements.
Transmission electron microscopy (TEM) and high-resolution TEM images were recorded on a
FEI TECNAI G2 SPIRIT TWIN operating at 120 kV and a JEOL JEM-2010F microscope
operating at 200 kV, respectively. The elemental composition of S10C5H QDs was analyzed
using energy-dispersive X-ray (EDX) spectroscopy. Prior to measurement, QDs were rinsed with
a 1:3 ratio of water-to-isopropanol and filtered using Amicon 3 kDa desalting filters (EMD
Millipore) to remove byproducts and oleic acid. Approximately 20 µL of the resulting solution
was placed on a nickel-coated TEM grid and dried under ambient conditions. Elemental
measurements were then made using a JEOL JEM-2010F microscope.
Fabrication of core/shell and its impact on the optical properties of QDs
Ternary I-III-VI semiconductor nanocrystals or quantum dots (QDs), such as near-infrared (NIR)
emitting copper indium selenide (CISe) QDs presented here, are attractive alternatives to the
more well-studied Cd-, Pb-, or Hg-containing QDs for in vivo imaging applications (27, 54). In
addition to avoiding potential toxicity from cadmium and lead ions, these QDs can also be
excited and emit in the NIR where penetration through biological tissue is favorable. However,
the PL QY of I-III-VI QDs cores is typically low (<10%). It has been demonstrated that coating
QDs with epitaxially-grown wide-bandgap semiconducting materials is a very efficient means to
electronically passivate the QD surface for boosting the PL QY and achieving better stability
against photobleaching (55-57). Zinc sulfide (ZnS), a non-toxic, stable, and abundant
semiconducting material, was chosen to overcoat the as-prepared CISe cores due to its large
bandgap (Eg,bulk 3.61 eV), which provides a type I band alignment with CISe (Eg,bulk 1.05
eV). Furthermore, the lattice mismatch between ZnS and CISe is around 7%, thereby enabling
the epitaxial growth of ZnS shell without introducing intensive strain on the cores and thus high
PL QY. In some QD formulations, aluminum ions were also introduced into ZnS lattice during
shell growth in order to further enhance long-term photostability against oxidation. It was
previously found Al doped in the CdS shell on CdSe cores formed into aluminum oxide, which
can serve as a self-passivation layer on the surface of the core/shell QDs and effectively enhance
photostability (50). Therefore, aluminum doped ZnS shells were designed to enhance the
resistance to photodegradation under harsh and/or long-term light irradiation.
Typical temporal evolutions of optical properties of CuInSe2 cores by using
stoichiometric ratio and shell growth are shown in Fig. 2, B and C, and figs. S2 and S3. In Fig.
2B and fig. S2A, a 1 h shelling reaction leads to an obvious blue shift of the PL emission peak
from 964 nm to 916 nm, and when the coating reaction was prolonged, the position of PL
emission only slightly changed (further blue-shifted to 891 nm after 5 h). The initial blue shift or
increase of the quantum confinement was resulted from the shrinkage in the emissive core size,
likely due to near-surface Zn interdiffusion. This cannot be attributed to the further growth or
ripening of the CISe cores, although unpurified CISe cores were directly used as seeds for further
shell growth in the presence of excess of CISe precursors. The spectroscopic observations in the
late stages of reaction strongly suggests that the distinct epitaxial shell coating process is
dominant over Zn2+ indiffusion, which serves as a protective barrier surrounding the core and
accounts for the further PL enhancement. As shown in Fig. 2C, 1 h of shelling time resulted in an
obvious increase in PL QY, (16.2% to 27.6%) when exited at 405 nm due to initial surface
passivation. Afterwards, PL QY further increased with prolonged shell growth time, reaching to
43.6% (λex: 405 nm) and 31.3% ((λex: 808 nm) after 5 h of shell growth.
CuInSe2 has the chalcopyrite structure which is a slightly modified form of zinc blende,
where Cu+ and In3+ ions occupy the Zn2+ positions, similar to the case of CuInS2 (51). Zn2+ ions
readily diffuse into the CuInSe2 lattice due to the small difference of ionic radii between Zn2+ and
Cu+. Therefore, the initial PL shifts can be attributed to the Zn2+ indiffusion into near-surface
lattice of CISe core. As a consequence, the radiative transition of the conduction band involving
Zn2+ to internal defect levels occurs. The formation of Zn graded core and etched cores prior to
surface epitaxial shell coating has been shown to account for a similar phenomenon during the
initial shelling stage of CuInS2 (51), resulting in both an increase the band gap of starting
fluorescent center due to the wider band gap of bulk ZnS than that of CISe. In the meantime, the
observed narrowing of PL full width at half maximum (FWHM) against shell growth suggests
that an annealing process of the CISe incorporated with Zn2+ ions and simultaneous ZnS shell
growth did not give rise to either size or composition distribution broadening for the particles in
the ensemble (fig. S2).
To further confirm the presence of the shell structure, the time-resolved PL (TRPL)
measurements were performed to reveal the nature and influence of various surface states
involved in the emission processes. The PL decay curves measured at the PL emission peak
max) are shown in fig. S3A. In general, the PL relaxations of the CISe seeds and their core/shell
QDs were characterized by multi-exponential processes that were best fit by three-exponential
decays (all decay fitting data are summarized in table S2). The average lifetime of the entire
fluorescence decay process was further determined from these three-exponential decay fits (fig.
S3 inset). The entire recombination process of the initial CISe core particles was comprised of
three decay components with time constants of 4.1 ns, 55.1 ns, and 218.5 ns as shown in table
S2. The origin of PL emission in ternary I-III-VI nanocrystals is not fully understood, but may
involve surface defect-related recombination, conduction band to internal defect recombination,
and donor-acceptor pair recombination processes. The fitting data suggest the first recombination
channels for CISe core emission (the surface defect related recombination) is partially
suppressed with its component amplitude (B1) decreasing from 35.0% to 26.5% after 5 h of
shelling. This suggests the shell coating suppresses the fast decay channel by inhibiting the
effects of the surface state on emission while recombination from the conduction band to internal
defects was enhanced with the component amplitude increasing from 22.8% to 34.6% (B2). The
lifetime associated with this fast decay slightly increases from 4.1 ns to 4.7 ns indicating a
reduction of the recombination rate via this channel and so the fast component contribution was
slightly decreased, going from 1.7% to 1.5% of the overall emitted fluorescence. A more
substantial impact of shelling is seen in the changes to the second decay process with regards to
the conduction band to internal defect recombination. Here the emission linked to this process
increases in both amplitude and lifetime, resulting in a net increase in PL emission from 14.9%
to 25.8%. This increase is seen as evidence of the aforementioned diffusion of Zn into the core
surface lattice, filling defects (vacancies) and thereby healing them. Furthermore, in contrast to
the second term, the third terms associated with donor-acceptor related recombination diminishes
in terms of lifetime as well as its net contributions to emission, from 252.1 ns to 183.2 ns, and
from 83.4% to 72.7%, respectively. This suggests that the Cu-related internal defects largely
accounting for the third slow recombination process were decreased by Zn interdiffusion to core.
As a result, the average lifetime presents a decreasing trend against shell growth time as shown
in the inset of fig. S3A. However, this recombination channel still dominated the whole emission
process, suggesting the nature of the emission of CISe core QDs was maintained and internal
defects at the origin of the PL emission are located inside the CISe cores.
The recombination rates derived from transient PL spectroscopy are plotted against shell
growth time in fig. S3B. A larger increase for radiative recombination rate is observed against
shell growth time, while the non-radiative recombination rate remained nearly unchanged. In
principle, if the radiative recombination rate is increased when forming a heterostructure, it is
necessary to suppress the increase in the non-radiative recombination rate to obtain high PL QY.
Therefore, the overgrowth of the shell with a wider band gap ZnS-based materials reported
herein effectively prevents exciton trapping at surface states, which are largely responsible for
the quality of resulting core/shell interfaces formed during the shelling reaction, thereby
presenting an increasing tendency in the ensemble PL QY against shell growth. In absolute
terms, these relative changes give rise to an overall improvement in the PL QY, from a core QD
PL QY of around 16.2% to a core/shell QD with lowered defect levels having a PL QY of
around 43.6%. All these variations support the presence of an outer ZnS shell structure proposed
above, with some indiffusion of Zn into the core. Therefore, the CISe core most likely transitions
to a Zn gradient structure before reaching a ZnS outer shell.
The PL decay curves were fitted using a multi-exponential function:
(S1)
In this expression,
i
represent the decay time constants, and
i
B
represents the normalized
amplitude of each component, n is the number of decay times. Because the photoluminescence
decays for all the QDs are best fitted using a two-/three-exponential model (n = 2/3), the
amplitude weighted average decay lifetime
avg
of the entire fluorescence decay process was
calculated with the form:
(S2)
The normalized lifetime-amplitude product is given as:
ii
ii
iB
B
f
(S3)
In this expression,
i
f
represents the relative time-integrated contribution of each respective
process to the overall number of emitted photons (i.e., the emission intensity measured in steady
state PL spectra).
In addition, given the experimentally determined PL quantum yield (QY), the radiative
recombination lifetime (
r
) is given as:
QY
avg
r
(S4)
and then the non-radiative recombination lifetimes (
nr
) is given as:
)/1/1( 1
ravg
nr
(S5)
The PL decay-fitting data for all curves are summarized in table S2.
Computational modeling of microneedle geometry
COMSOL Multiphysics was used to perform finite element analysis on various microneedle
shapes. Microneedle structure was numerically modeled using a linear elastic constitutive
material model with an elastic modulus of 1.7 GPa in accordance with data obtained from tensile
testing experiments (1.7 ± 0.3 GPa, n = 8) and Poisson's ratio of 0.45. The Structural Mechanics
Module was used to run simulations with a physically-controlled, extra fine mesh size. The
geometries explored included a cone, cylinder, and intermediate portions thereof in which the
lower portion of the microneedle was a cylinder and the upper portion of the microneedle was a
cone. Microneedle height was fixed at 1500 µm. As a result, the shape could be fully defined by
two parameters: microneedle diameter and alpha, which represented the proportion of the
microneedle height that was cylindrical.
Fifty distinct microneedle designs with microneedle diameter ranging from 100 µm to
300 µm and alpha varying from 0 to 0.9 were evaluated for six criteria: critical load factor under
buckling, displacement under axial loading, von Mises stress under axial loading, displacement
under bending, von Mises stress under bending, and theoretical deliverable volume at the
microneedle tip. Buckling measurements were performed based on a method previously reported
1 )(exp )( 11
n
iii
n
iiB-t/τBtI
(30). Briefly, the microneedle tip was fully constrained and a total downward load of 5 N was
applied to the base center while the microneedle was free to move only along the shaft axis. To
perform axial loading simulations, a pressure of 3.18 MPa, equal to the skin penetration stress
(58), was applied to the tapered region in an axial direction (along the shaft). Bending
simulations were conducted by applying a total lateral load of 20 mN to the tapered region,
similar to a method previously reported (59). All degrees of freedom of the microneedle base
were fully constrained in both axial and bending loading analyses. Maximum von Mises stress
and maximum displacement in the structure were obtained from axial and bending load analysis
while critical buckling loadrepresenting the stability of structure against bucklingwas
calculated from buckling analysis. Based on these analyses, we generated dissolvable needles
with favorable properties for skin penetration and deliverable volume.
Microneedle mold fabrication
Microneedle arrays were designed using Sketchup Make (Trimble Inc.) and exported as
stereolithography (.stl) files. These computer-aided design files were then opened in DeScribe
(Nanoscribe GmbH), sliced, and hatched using adaptive slicing with a minimum slicing distance
of 0.1 µm, shell and scaffold printing with a 1 µm hatching distance, shell contour count of 6,
base slice count of 12, and 1 µm hatching distance with a triangular interior scaffold and power
setting of 50,000. The exported files were then loaded into NanoWrite and printed using a
Nanoscribe to perform dip-in laser lithography with a 25x objective lens in IP-S atop glass
coated with indium tin oxide. After the print had completed, unexposed IP-S was removed by
soaking the slide in propylene glycol methyl ether acetate for 10 min and then soaking in
isopropanol for 10 additional min.
Photoresist master molds were then coated with a thin layer of trichloro(1H,1H,2H,2H-
perfluorooctyl) silane by placing the mold alongside a glass slide covered with the chemical in a
desiccator under house vacuum for 1 hour. The mold was then fixed to the bottom of a petri dish
using double-sided tape and covered with polydimethylsiloxane (PDMS, Sylgard 184, Dow
Corning) mixed according to the manufacturer’s instructions, and placed overnight in an oven at
60°C. The cross-linked PDMS was then removed from the photoresist mold and subsequently
used to create mater mold replicates or dissolvable microneedles. To create additional mater
molds, the UV-curable Norland Optical Adhesive 61 was filled into the PDMS molds using a
centrifuge at 3,234 RCF for 5 min placed in a UV-curing oven at room temperature for 15 min
and manually removed by attaching a small piece of glass to the backing of the patch using
double-sided tape.
Mechanical testing of microneedles
The force required to penetrate synthetic human skin (SynDaver) or explanted porcine skin was
evaluated using a Dynamic Mechanical Analyzer Q800 (TA Instruments) in compression mode.
Microneedle arrays composed of Norland Optical Adhesive 61 (Norland Products) were used to
prevent complications due to dissolution. Microneedle patches were inverted and affixed to the
top platform of the compression clamp using double-sided tape while skin was placed on the
bottom compression platform. An initial pre-load force of 2.5 mN was applied to bring the
needle tips into contact with the skin. Force was then applied to determine the amount required
to penetrate the skin surface. Visual inspection was used rather than displacement because of the
propensity of the skin sample to compress. A minimum force step of 2.5 mN was used to
determine the minimum penetration force required. 3x3 arrays of each microneedle geometry
tested were analyzed using three trials each in different areas of the skin sample. Studies
evaluating the effect of needle spacing utilized 3x3 arrays of the optimized microneedle shape
(750/750 µm cylinder/cone) at a spacing of 900 µm or 3233 µm.
In vitro evaluation of quantum dot toxicity
The in vitro toxicity of QDs with a copper indium selenide core and aluminum-doped zinc
sulfide shell was evaluated in mouse macrophages (Raw 264.7 cells) and compared to PMMA-
encapsulated QDs and commercial lead sulfide (PbS) core-type QDs with an oleic acid coating
(Sigma-Aldrich). Prior to plating cells, samples were plated onto 96-well glass bottom plates
(Cellvis) in 100 µL of either toluene for hydrophobic unencapsulated QDs or sterile water for
PMMA-encapsulated QDs at 1000, 200, 40, and 8 µg/ml. After drying, cells were plated at
10,000 cells/well in 100 µL of phenol red-free DMEM (Life Technologies) supplemented with
10% fetal bovine serum and 1% penicillin/streptomycin and incubated at 37°C in 5% CO2 for 24
hours. Cell viability was then determined using a CellTiter-Glo Luminescent Assay (Promega)
according to the manufacturer’s instructions.
Ex vivo microneedle application
Microneedle penetration and microparticle delivery was assessed ex vivo in Yorkshire pig skin
obtained from the ear and human cadaveric skin donated to the National Disease Research
Interchange fulfilled under protocol DLAR9-001. Microneedle patches were imaged before and
after administration using a Leica M80 microscope and Leica IC80 HD Camera and scanning
electron microscope using the preparation and imaging parameters described above. Tissue
samples were also imaged before and after the application of a dissolvable microneedle patch
containing PMMA-encapsulated QDs using the adapted smartphone with and without LED
illumination and emission filters.
Image processing
After collection, images were downloaded from the phone, cropped to a size of 237x279 in
Adobe Photoshop to isolate a single pattern, and saved as portable network graphics (.png) files.
These images were then used directly for detection via machine learning. For signal
quantification in longitudinal imaging studies, cropped images were further split into three
constituent red, green, and blue channels in ImageJ and the red channel was saved as a grayscale
image. For signal-above-background quantification in the short-terms study, circular ROIs 80
pixels in area were then manually placed for each of the sixteen markings in the 4x4 array. The
average value of the remaining pixels in the image was then subtracted to obtain signal-above-
background. SNR was calculated similarly; however, to reduce diffuse light surrounding the NIR
markings, which would have a larger effect on SNR, background was calculated by measuring
the average intensity of all pixels that fell outside 616-pixel ROIs associated with each marking
and then taking the average of all pixels outside of this area. This technique allowed for a
compromise between adequately representing the brightness of the signal while eliminating most
diffuse, signal-associated brightness that is not representative of the true background. The signal
from each marking was then divided by the background signal to generate a SNR. Using smaller
signal ROIs or larger ROIs for background subtraction would serve to further increase SNR.
Machine learning detection algorithm
Transfer learning-based machine learning has recently emerged as a promising method for image
classification, particularly when there is a lack of sufficient training data (60, 61). Here, we
applied this approach to a pre-trained AlexNet neural network, a classification tool with 25
layers (20) tuned to classify three spatial patterns of interest: a circle, cross, and rectangle. The
Deep Learning Toolbox model of AlexNet available in MATLAB was used for all image
classification. The first 22 layers of AlexNet (1-22), which contained the image segmentation
and feature extraction properties, were directly transferred from the original AlexNet. The top
three layers (22-25) were modified to classify the three patterns of interest.
To generate a training dataset, 30 images per pattern were selected from animals not
included in the test data, which will subsequently be referred to as source images. Image
augmentation was then performed on each source image containing an individual pattern by
applying a set of modifications using a pre-processing code (62). This code performed a series of
functions including image cropping, inversion, and rotation, brightness normalization, grayscale
conversion, and resizing. The set of 30 modified source images per pattern were each then used
to generate 100 augmented image for a total of 3,000 images per pattern (9,000 total). Using this
approach, 70% of the generated images were used for training the last three layers of the network
while 30% were retained for validation. Rectified Linear Unit (ReLu) was employed as the
activation function. A maximum epoch number of 20, minibatch size or 10, and initial learning
rate of 1x10-4 were selected for training. The training approach was based on labeled and
supervised data and resulted in a validation accuracy of 100% after the twentieth epoch (fig. S8).
Training was performed on a single Nvidia GeForce GTX1080 graphics processing unit and was
completed in approximately 2-3 hours.
The trained neural network was then used to classify images from the long-term
experimental group receiving QD-loaded microparticles from dissolvable microneedle patches.
Images were modified using the same pre-processing method used above prior to their analysis.
The classification output consisted of a 3x1 array representing the relative likelihood that the
image belonged to each of the trained patterns (circle, cross, or the rectangle). This value was
reported as the probability of each classification.
Supplementary Figures:
Fig. S1. Optical properties of organic dyes. (A) Emission profiles of NIR organic
fluorophores. (B) PL QY of NIR organic dyes considering only emission above 850 nm upon
excitation at 780 nm.
Fig. S2. Evolution of fluorescence emission properties with shelling time. Graphs showing
the change in PL emission peak maximum max) with shelling time (top), full width at half
maximum (FWHM) of PL peaks (bottom, squares), and FWHM/λmax (bottom, triangles) for the
S10C series of QDs, which includes S10C5H, the QD used for all in vivo studies.
Fig. S3. Fluorescence lifetime characterization of the S10C QD series. (A) Normalized time-
resolved PL decay curves together with (inset) corresponding time-resolved PL average lifetimes
and (B) radiative (purple circles) and non-radiative (green squares) recombination rates as a
function of shell growth time for S10C series.
Fig. S4. Composition and physical properties of S10C5H QDs. (A) Energy-dispersive X-ray
spectrum and annotated atomic composition of S10C5H QDs. Ni was detected due to the TEM
grid substrate. (B) Selected area electron diffraction (SAED) pattern of S10C5H QDs. The well-
resolved SAED pattern confirm the chalcopyrite (tetragonal) phase structures, which is the stable
phase of bulk CuInSe2 at room temperature (JCPDS 40-1487). (C) TEM size histogram of
S10C5H QDs. (D) Hydrodynamic size histogram of S10C5H QDs dispersed in dichloromethane.
Fig. S5. pH stability of PMMA-encapsulated QDs. The fluorescence intensity of encapsulated
S10C5H QDs was compared before and after 22 hours of incubation at pH 4 to 10. n = 4-5, P >
0.05 for all pH values tested using a two-way ANOVA with Tukey’s multiple comparisons test.
Fig. S6. Finite element analysis of mechanical forces on microneedles. (A) Microneedle shape
was modeled in silico with shapes ranging from a cone to a cylinder. (B) Finite element analysis
was used to assess the mechanical forces applied during microneedle penetration. Distribution of
(C) von Mises stress during bending, (D) displacement during bending, (E) von Mises stress
under axial load, (F) displacement under axial load, and (G) total displacement for the optimized
design (alpha = 0.5, D = 300 µm).
Fig. S7. Optimization of microneedle geometry using finite element analysis. FEA-based
optimization of the microneedle geometry was performed regarding six criteria based on
microneedle diameter and alpha (the proportion of microneedle height that was cylindrical). (A)
maximum von Mises stress under axial loading, (B) maximum displacement under axial loading,
(C) maximum von Mises stress under bending, (D) maximum displacement under bending, (E)
critical load factor under buckling, and (F) theoretical deliverable drug volume, calculated as the
volume of top half of the solid microneedle. In total, 50 geometries were evaluated with shaft
diameter ranging from 100 to 300 µm, and alpha ranging from 0 to 0.9.
Fig. S8. Machine learning training and validation. Training and validation results of the
modified AlexNet network. The resulting validation accuracy after 20 epochs was obtained as
100%, and the loss went to 0.
Supplementary Tables:
Table S1. Spectral characterization of custom QD formulations. The effect of quantum dot composition and shelling time on
emission wavelength and quantum yield. Formulations selected for further evaluation highlighted in red.
Formulation
PL
Emission
Peak (nm)
PL
FWHM
(nm)
FWHM
/ Peak
PLQY at
405nm (%)
Cu:In:Se
(mol:mol)
[Cu]
mol/L
Cu:DDT
(mol:mol)
DDT:OLA:OA:
ODE (mL:mL)
Zn:Al
(mol:mol)
Shelling
Time
S2C1.5H
981
187
0.19
37.1±0.1
1:1:2
0.038
1:12
1:0.67:0.33:6.67
1:0
1.5 h
S2C2H
981
175
0.18
35.4±0.1
2 h
S3C2H
981
181
0.18
31.0±0.2
1:1:2
0.038
1:12
1:0.67:0.33:6.67
1:0.3
2 h
S4C1H
750
135
0.18
28.3±0.1
1:4:10
0.013
1:67
1:0.6:0.06:3
1:0.3
1 h
S4C2H
750
141
0.19
36.5±0.1
2 h
S5C0.5H
835
157
0.19
29.5±0.2
1:2:6
0.021
1:40
1:0.6:0.06:3
1:0.3
0.5 h
S5C1H
840
151
0.18
28.3±0.1
1 h
S5C2H
820
145
0.18
36.5±0.1
2 h
S5C3H
810
143
0.18
38.1±0.1
3 h
S8C0H
938
145
0.15
24.2±0.2
1:1.5:2.5
0.029
1:23
1:0.43:0.21:4.29
1:0.3
0 h
S8C1H
895
129
0.14
29.3±0.1
1 h
S8C2H
886
128
0.14
33.9±0.2
2 h
S8C5H
860
127
0.15
41.9±0.2
3 h
S9C0H
1036
244
0.24
10.3±0.0
1:1:2
0.036
1:19
1:0.43:0.21:4.29
1:0
0 h
S9C0.5H
1022
237
0.23
22.5±0.1
0.5 h
S9C1H
1010
245
0.24
9.0±0.03
1 h
S9C3H
1009
219
0.22
21.1±0.1
3 h
S9C5H
968
233
0.24
30.7±0.0
5 h
S10C0H
964
168
0.17
16.2±0.3
1:1:2
0.038
1:12
1:0.67:0.33:6.67
1:0.3
0 h
S10C0.5H
954
159
0.17
37.2±0.1
0.5 h
S10C1H
916
157
0.17
27.6±0.2
1 h
S10C2H
907
152
0.17
30.0±0.2
2 h
S10C3H
897
146
0.16
33.6±0.1
3 h
S10C5H
891
145
0.16
43.6±0.1
5 h
S11C0H
952
141
0.15
26.6±1.3
1:1.5:2.5
0.031
1:15
1:0.67:0.33:6.67
1:0.3
0 h
S11C0.5H
916
134
0.15
38.7±0.4
0.5 h
S11C1H
906
129
0.14
35.4±1.1
1 h
S11C2H
888
124
0.14
40.4±0.6
2 h
S11C3H
879
121
0.14
58.8±2.6
3 h
S11C5H
858
123
0.14
48.7±0.6
5 h
S14C0H
766
132
0.17
21.3±0.6
1:2:3
0.026
1:18
1:0.67:0.33:6.67
1:0
0 h
S14C1H
724
126
0.17
22.2±0.1
1 h
S14C2H
696
124
0.18
37.8±4.8
2 h
S14C3H
680
124
0.18
34.3±0.1
3 h
S14C5H
664
110
0.17
38.3±0.2
5 h
S15C0H
750
131
0.17
19.0±0.3
1:2:3
0.025
1:28
1:0.43:0.09:4.29
1:0
0 h
S15C1H
704
129
0.18
29.1±0.3
1 h
S15C5H
634
145
0.23
42.6±0.1
5 h
S16C0H
880
135
0.15
38.7±0.4
1:2:3
0.024
1:28
1:0.43:0.21:4.29
1:0
0 h
S16C0.5H
878
136
0.15
32.8±0.6
0.5 h
S16C1.5H
860
125
0.15
31.5±1.0
1 h
S16C2H
858
127
0.15
34.6±0.5
2 h
S16C3H
838
130
0.16
37.5±0.2
3 h
S16C5H
812
122
0.15
43.0±0.8
5 h
S17C0H
964
169
0.18
17.3±1.0
1:1:2
0.036
1:19
1:0.43:0.21:4.29
1:0
0 h
S17C0.5H
954
164
0.17
32.5±1.5
0.5 h
S17C1H
940
163
0.17
35.5±0.1
1.5 h
S17C2H
910
160
0.18
38.3±0.9
2 h
S17C3H
902
153
0.17
35.4±0.1
3 h
S17C5H
902
143
0.16
48.4±0.3
5 h
S18C0H
958
167
0.17
15.4±0.1
1:1:2
0.038
1:12
1:0.67:0.33:6.67
1:0.5
0 h
S18C0.5H
940
152
0.16
33.2±1.1
0.5 h
S18C1H
918
147
0.16
30.6±0.3
1 h
S18C2H
904
148
0.16
32.6±0.8
2 h
S18C3H
894
141
0.16
29.6±0.3
3 h
S18C5H
884
139
0.16
31.4±0.2
5 h
S18C7H
886
140
0.16
31.6±0.1
7 h
S18C18H
878
138
0.16
34.9±0.0
18 h
S19C0.5H
956
158
0.17
34.6±0.1
1:1:2
0.038
1:12
1:0.67:0.33:6.67
1:0.5
0.5 h
S19C1H
940
159
0.17
28.6±0.3
1 h
S19C2H
920
163
0.18
28.1±0.2
2 h
S19C3H
914
150
0.16
25.7±0.0
3 h
S19C5H
900
145
0.16
28.3±0.2
5 h
S19C7H
886
136
0.15
40.3±0.1
7 h
S19C19H
872
133
0.15
42.0±0.1
19 h
S19C24H
866
136
0.16
41.8±0.2
24 h
S19C48H
874
136
0.16
10.3±0.2
48 h
S19C72H
865
140
0.16
9.1±0.0
72 h
S20C0H
922
136
0.15
23.7±0.7
1:1.5:2.5
0.029
1:23
1:0.43:0.21:4.29
1:0.3
0 h
S20C0.5H
878
127
0.14
25.9±0.4
0.5 h
S20C1H
866
127
0.15
21.3±0.1
1 h
S20C2H
856
126
0.15
31.5±0.5
2 h
S20C3.5H
846
129
0.15
35.9±0.9
3.5 h
S20C5H
828
133
0.16
35.1±0.5
5 h
Note: PL and PL QY data for the S12C and S13C QD series are distorted and likely underestimated due to absorption by the solvent (cyclohexane).
Table S2. Multiexponential fitting parameters for photoluminescence decay curves. The
parameters for multi-exponentially fitting the photoluminescence decay curves in fig. S3,
normalized amplitude Bi, time constant
i and their normalized products
i
f
, goodness-of-fit
parameter
², together with the detection wavelength.
Sample
em a
[nm]
QY b
[%]
B1
[%]
f1
[%]
1
[ns]
B2
[%]
f2
[%]
2
[ns]
B3
[%]
f3
[%]
3
[ns]
avg
[ns]
²
0 h (Core)
964
16.2
35.0
1.7
4.1
22.8
14.9
55.1
27.9
83.4
252.1
218.5
0.9992
1 h
916
27.6
26.5
1.4
4.9
33.8
24.2
68.1
33.4
74.4
211.6
174.0
0.9995
2 h
907
30.0
23.8
1.5
5.9
38.9
30.8
74.5
30.5
67.7
209
164.5
0.9995
3 h
897
33.6
27.8
1.3
4.1
33.7
24.3
60.8
32.9
74.4
191
156.9
0.9996
5 h
891
43.6
26.5
1.5
4.7
34.6
25.8
59.7
31.8
72.7
183.2
148.6
0.9997
a emission peak position, b excitation at 405 nm
Supplementary Movies:
Movie S1. Intradermal administration and imaging of encapsulated QDs. Prior to filming
rats were anesthetized and a small area of the flank was shaved, treated with depilatory cream,
and sterilized using an ethanol swab. The experimental setup included a 780 nm LED
illumination source focused on the shaved area of the rat, a NIR-adapted smartphone, a spring-
loaded microneedle applicator, and a QD-loaded microneedle patch. Prior to application, no NIR
signal was visible on the rat, but was apparent in the microneedle patch. The microneedle patch
was then applied for two minutes using the spring-loaded applicator, which transferred QDs into
the skin due to dissolution of the microneedle matrix. The transferred QDs were then viewed
using the NIR-adapted smartphone to reveal a 4x4 square array.
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Here we describe a new mouse model that exploits the pattern of expression of the high-affinity IgG receptor (CD64) and allows diphtheria toxin (DT)–mediated ablation of tissue-resident macrophages and monocyte-derived cells. We found that the myeloid cells of the ear skin dermis are dominated by DT-sensitive, melanin-laden cells that have been missed in previous studies and correspond to macrophages that have ingested melanosomes from neighboring melanocytes. Those cells have been referred to as melanophages in humans. We also identified melanophages in melanocytic melanoma. Benefiting of our knowledge on melanophage dynamics, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture–release–recapture without any tattoo vanishing. Therefore, congruent with dermal macrophage dynamics, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity.
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Background In 2007–08, a genotype J mumps outbreak occurred among Aboriginal people in northern Western Australia, despite high vaccine coverage. In March, 2015, a second protracted mumps outbreak occurred in northern Western Australia and spread widely across rural areas of the state. This time the outbreak was caused by a genotype G virus and again primarily affected Aboriginal people. We aimed to describe the epidemiology of this outbreak. Methods In this population-based surveillance study, we analysed statutory notifications and public health case follow-up data from the Western Australia Notifiable Infectious Diseases Database and vaccination information from the Australian Childhood Immunisation Register. An outbreak case of mumps was notified if the affected person was living in or visiting a community in Western Australia where there was active mumps transmission, and if mumps infection was confirmed by laboratory diagnosis or by an epidemiological link. We analysed case demographics, vaccination status, and age-standardised attack rates in Aboriginal and non-Aboriginal people by region of notification. Laboratory diagnoses were made by real-time RT-PCR, serology, or both, and carried out by the sole public pathology provider in Western Australia. Findings Between March 1, 2015, and December 31, 2016, 893 outbreak cases were notified. 798 (89%) of 893 outbreak cases were reported in Aboriginal people. 40 (4%) of 893 people were admitted to hospital, and 33 (7%) of 462 men reported orchitis. Mumps attack rates increased sharply with age, peaking in the 15–19 age group. 371 (89%) of 419 people aged 1–19 years were fully vaccinated and 29 (7%) were partly vaccinated. Of the 240 people who tested positive by real-time RT-PCR and had also been tested for mumps-specific IgG and IgM, 165 (69%) were positive for IgG but negative for IgM, indicating the importance of RT-PCR testing for diagnosis in vaccinated populations. None of the cases from the 2007–08 genotype J outbreak were re-notified. Interpretation The number of mumps outbreaks reported in recent years among highly vaccinated populations, including Indigenous populations, has been growing. More widespread and pre-emptive use of the third dose of measles, mumps, and rubella vaccine might be required to control and prevent future outbreaks in high-risk populations. Research should explore the benefit of increasing the intervals between vaccine doses to strengthen the durability of vaccine protection.
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The utility of layer-by-layer (LbL) coated microneedle (MN) skin patches for transdermal drug delivery has been proven a promising approach, with advantages over hypodermal injection due to painless and easy self-administration. However, the long epidermal application time required for drug implantation by existing LbL MN strategies (15 to 90 minutes) can lead to potential medication noncompliance. Here, we developed a MN platform to shorten the application time in MN therapies based on a synthetic pH-induced charge-invertible polymer poly(2-(diisopropylamino) ethyl methacrylate-b-methacrylic acid) (PDM), requiring only 1-minute skin insertion time to implant LbL films in vivo. Following MN-mediated delivery of 0.5 μg model antigen chicken ovalbumin (OVA) in the skin of mice, this system achieved sustained release over 3 days and led to an elevated immune response as demonstrated by significantly higher humoral immunity compared with OVA administration via conventional routes (subcutaneously and intramuscularly). Moreover, in an ex vivo experiment on human skin, we achieved efficient immune activation through MN-delivered LbL films, demonstrated by a rapid uptake of vaccine adjuvants by the antigen presenting cells. These features—rapid administration and the ability to elicit a robust immune response—can potentially enable a broad application of microneedle-based vaccination technologies.
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Approximately 1.7 million new cases of cancer will be diagnosed this year in the United States leading to 600 000 deaths. Patient survival rates are highly correlated with the stage of cancer diagnosis, with localized and regional remission rates that are much higher than for metastatic cancer. The current standard of care for many solid tumors includes imaging and biopsy with histological assessment. In many cases, after tomographical imaging modalities have identified abnormal morphology consistent with cancer, surgery is performed to remove the primary tumor and evaluate the surrounding lymph nodes. Accurate identification of tumor margins and staging are critical for selecting optimal treatments to minimize recurrence. Visible, fluorescent, and radiolabeled small molecules have been used as contrast agents to improve detection during real-time intraoperative imaging. Unfortunately, current dyes lack the tissue specificity, stability, and signal penetration needed for optimal performance. Quantum dots (QDs) represent an exciting class of fluorescent probes for optical imaging with tunable optical properties, high stability, and the ability to target tumors or lymph nodes based on surface functionalization. Here, state-of-the-art biocompatible QDs are compared with current Food and Drug Administration approved fluorophores used in cancer imaging and a perspective on the pathway to clinical translation is provided.