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Representative images of hairless rat skin in vivoduring recovery after hGH administration. Each group is the same group as in Figure S5.The skin was occluded for the first 24 h and unoccluded afterwards.Figure S2. Skin erythema rating after treatment withmicroneedles. Erythema scale: 0 = normal skin, 1 = slight, 2 =mild, 3 = moderate, and 4 = severe erythema. Each group is the same asdefined in Figure S5.
Skin erythema was not rated during the first 24 hours, because itwas occluded and could not be seen. No edema was seen in any groups. Data pointsrepresent the average ± standard deviation for n ≥ 4 for up to 72hours.Figure S3. Representative histological images of skin biopsiedfrom hairless rats in vivo. Each group is the same as described in Figure S5. At 24 h,Groups 2 and 4 show the sites of microneedle insertion (arrows) and appear to undergohighly localized inflammatory responses (see Figure S4 for magnified views).Figure S4. Representative histological images under highmagnification of skin biopsied from hairless rats in vivo.
(A) Normal skin (negativecontrol). (B) Subcutaneously injected skin (Group 1). Skin at sites of microneedleinsertion 24 h after treatment with a (C) CMC microneedle patch (Group 2) and (D)CMC/trehalose microneedle patch (Group 4). Note the presence of cells at the sites ofinsertion in (C) and (D) (arrows), which appear to be associated with inflammation.Figure S5. Summary of hGH formulation and administration. Group1: subcutaneous injection of hGH, Group 2: CMC microneedle patch encapsulating hGH,Group 3: subcutaneous injection of reconstituted CMC microneedle patch encapsulating hGHafter 15 months storage at ambient condition, Group 4: CMC/trehalose microneedle patchencapsulating hGH, and Group 5: subcutaneous injection of reconstituted CMC/trehalosemicroneedle patch encapsulating hGH.
Clinical impact of biotechnology has been constrained by the limitations oftraditional hypodermic injection of biopharmaceuticals. Microneedle patches have beenproposed as a minimally invasive alternative. In this study, we assess the translation ofa dissolving microneedle patch designed for simple, painless self-administration ofbiopharmacetucials that generates no sharp biohazardous waste. To study pharmacokineticsand safety of this approach, human growth hormone (hGH) was encapsulated in 600 μmlong dissolving microneedles composed of carboxymethylcellulose and trehalose using anaqueous, moderate-temperature process that maintained complete hGH activity afterencapsulation and retained most activity after storage for up to 15 months at roomtemperature and humidity. After manual insertion into the skin of hairless rats, hGHpharmacokinetics were similar to conventional subcutaneous injection. After patch removal,the microneedles had almost completely dissolved, leaving behind only blunt stubs.
Thedissolving microneedle patch was well tolerated, causing only slight, transient erythema.This study suggests that a dissolving microneedle patch can deliver hGH and otherbiopharmaceuticals in a manner suitable for self-administration without sharp biohazardouswaste. IntroductionThe impact of biotechnology on medicine has been limited by the need for hypodermicinjection of protein therapeutics and other macromolecular drugs.The large molecular size and enzymatic sensitivity of these drugs precludes oraladministration, conventional transdermal delivery or absorption via mucosal routes in mostcases. Thus, many patients need to visit the clinic for injection by medical personnel whichis an inefficient use of resources.
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Alternatively, patients must be trained to injectthemselves, which requires a significant initial investment of time and brings the risks ofbiohazardous sharp needle waste into patient households and requires many patients to overcome theirfear and apprehension of hypodermic needles. , For example, children with short stature or other endocrine disorders require humangrowth hormone (hGH), which is injected subcutaneously multiple times per week for months toyears.
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As mentioned above, the major clinical problemassociated with hGH injection is patient non-compliance or refusal to inject due toneedle-phobia. Amuch better way to administer hGH would enable self-administration without special training,eliminate pain and apprehension, and avoid the generation of sharp, biohazardous waste.Previous attempts to administer hGH without a hypodermic needle, for example usingconventional transdermal delivery or intranasal delivery, have had very lowbioavailability. Here, we present the translation of adissolving microneedle patch designed for simple, painless self-administration of hGH thatgenerates no sharp biohazardous waste.Microneedle patches have been developed to combine the convenience and safety oftransdermal patches with the hypodermic needle's ability to delivermacromolecules. These microneedles measure hundreds of micronslong, which is sufficient to cross the skin's permeability barrier of stratumcorneum , but isshort enough to avoid causing pain. Previous generations of microneedles have been made ofnon-water-soluble materials such as silicon, metals, and organic polymers and have been usedeither to pierce the skin to increase skin's permeability or prepared with a drugcoating that dissolves off upon insertion into skin.Initial efforts to encapsulate drugs within microneedles required harsh processingconditions incompatible with sensitive biomolecules. Recently, we and others have developedmicroneedles made of water-soluble materials that dissolve in the skin and thereby leave nosharp biohazardous waste.In this study, we seek to utilize our dissolving microneedle technology to assessits translation into a patch for administration of hGH.
We selected carboxymethylcellulose(CMC) and trehalose as water-soluble matrix materials of the microneedle patch, both ofwhich are safely used in the body in FDA-approved formulations. CMCwas included in the formulation to provide mechanical strength and trehalose was added toincrease microneedle dissolution rate. Using this approach, this study presents hGH deliveryusing a dissolving microneedle patch designed for simple, painless self-administration thatgenerates no biohazardous sharp waste.
By addressing questions about hGH stability,bioavailability and safety, we seek to translate the use of dissolving microneedle patchesfor eventual clinical administration of hGH, as well as other protein therapeutics. Fabrication of dissolving microneedle patch encapsulating hGHDesign and fabrication of a dissolving microneedle patch for hGH administrationrequired an interdisciplinary approach involving microfabrication and pharmaceutics inorder to enable gentle encapsulation, self-administration, effective delivery into skin,and safe disposal. We first prepared a master microneedle structure using UV lithographyand reactive ion etching, as shown in.The master structure was then used to cast an inverse mold out of polydimethylsiloxane(PDMS). To facilitate reliable insertion into skin, each microneedle was designed to be600 μm long with a tip radius measuring less than 10 μm. To providesufficient mechanical strength, microneedles tapered down to a base measuring 300μm wide. Dissolving microneedles patch.
Scanning electron micrograph of (A) an array ofmicroneedles and (B) a further magnified view of a single microneedle in a masterstructure microneedle patch. Functional activity of hGHTo assess biocompatibility of the fabrication processes, the stability of hGHencapsulated in dissolving microneedle patches was assessed by an established cellproliferation assay. As shown in, exposure of Nb2 cells to unprocessed hGH (positivecontrol) stimulated cell growth as a function of hGH concentration. Addition of CMC tounprocessed hGH stimulated cell growth equally well, which indicates that CMC had nodeleterious effect on hGH activity or cell proliferation. As a negative control,reconstitution of CMC microneedles (containing no hGH) and incubation with Nb2 cells didnot stimulate cell growth, further indicating that CMC was inert.
HGH stability after encapsulation in a dissolving microneedle patch. The addition of hGHto Nb2 cell culture in the stationary phase stimulated the proliferation of Nb2 cells,which was recorded at 3 days after hGH treatment and used as a measure of the functionalactivity of hGH after encapsulation in dissolving microneedles. Five experimental groupswere studied: CMC solution by the reconstitution of a placebo CMC microneedle patch (CMC,negative control, ●), hGH solution (hGH, positive control, ▲), hGHsolution mixed with CMC reconstituted from a placebo CMC microneedle patch (hGH +CMC, ▪), hGH and CMC solution reconstituted from a CMC microneedle patchencapsulating hGH (hGH in CMC microneedle patch, ◆), and hGH and CMC solutionreconstituted from a CMC microneedle patch encapsulating hGH after storage for 3 months atambient conditions (hGH in CMC microneedle patch (3 months), ▾). All groupscontain CMC and hGH at the same mass ratio (5 hGH: 95 CMC) at all hGH concentrations,except the positive control. Asterisk indicates comparison with the hGH positive control(two-way ANOVA, p 0.05 for. and p. Pharmacokinetics of hGH administered by dissolving microneedle patchThis study hypothesized that a dissolving microneedle patch can administer hGHvia the skin after insertion and dissolution of the microneedle matrix.
To assess this, weadministered hGH via five different methods and measured plasma concentration of hGH overtime in the hairless rat.As shown in, hGH administered byany of the methods quickly rose to a peak hGH plasma concentration at t max≈ 0.7 h (ANOVA, p 0.05) and then sharply decreased over the next 6 hourswith a half-life of t ½ ≈ 1.1 h (ANOVA, p 0.05). Although the shape of the pharmacokineticprofile was the same by each method, the absolute hGH concentrations, and hencebioavailability differed. Pharmacokinetic profile of hGH in rat serum after hGH administration using microneedles.Five experimental groups were studied to assess different methods of hGH administration:(1) subcutaneous injection (●), (2) CMC microneedle patch (▲), (3)subcutaneous injection of reconstituted CMC microneedle patch after 15 months storage atambient conditions (▾), (4) CMC/trehalose microneedle patch (◆), and (5)subcutaneous injection of reconstituted CMC/trehalose microneedle patch (▪). Datarepresent the average ± standard deviation, n=6 for all groups exceptGroup 3 (n=3).Significantly different from Group 1 (Student's t-test, p 0.05) and bioavailability (Student'st-test, p 0.05) indistinguishable from the positive control, which is consistentwith our in vitro finding that the process of hGH encapsulation does not damage hGHbioactivity.We next studies hGH administration using microneedle patches prepared with twodifferent formulations. The first formulation contained only CMC as the microneedlematerial.
In this case, plasma hGH concentration was significantly lower than the positivecontrol, corresponding to a peak hGH concentration, C max, that was 20%of the positive control (Student's t-test, p. Scanning electron micrographs of dissolving microneedles after 24 h insertion intohairless rat skin in vivo/.
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(A) CMC microneedle patch. (B) CMC/trehalose microneedlepatch.This confirmed the expectation that CMC/trehalose microneedles were able todissolve more completely and provides an explanation for the reduced bioavailability usingmicroneedles. In future studies, microneedle design could be modified to increaseinsertion depth more fully into the skin, to incorporate a more water-soluble formulationor to localize hGH more toward the tip and away from the base.
These, or othermodifications, could increase hGH bioavailability using microneedles.The final group in our pharmacokinetics study was designed to assess functionalstability of microneedles during storage. We reconstituted hGH from a CMC microneedlepatch after 15 months storage at room temperature and humidity and injected itsubcutaneously, which produced a bioavailability of 61±29%. Thisremarkable stability may be due to storage of hGH by coupling to the rigid glassymicroneedle matrix ,which limits molecular mobility and contact with oxygen from the air. In contrast,commercially formulated hGH is lyophilized and must be stored under refrigeration withouthumidity or oxygen, both of which lead to degradation of hGH integrity. Packaging microneedles withouthumidity or oxygen could further stabilize hGH during storage, as would storage at reducedtemperature.
Skin resealing after microneedle administrationIn this study, dissolving microneedles were used to pierce the skin barrier anddeposit hGH in the skin. Previous studies have shown that skin reseals quickly afterinsertion of non-dissolving microneedles.
We therefore determined the kinetics of skin resealing afterinsertion of dissolving microneedles using skin electrical impedance as a measurementpreviously shown to correlate with the skin's permeabilitybarrier. As shown in, impedance ofuntreated skin was initially around 10 5 kΩcm 2. Inthe negative control (Group A), which was untreated, but covered continuously for 24 hwith a measurement electrode, skin impedance dropped to less than 10 2kΩcm 2 and remained low due to the known effect of increasedskin hydration due to extended occlusion by the electrode. After the occluding electrode was removedfrom the skin at 24 h, skin impedance returned to original levels because the electrodewas only applied briefly at the time of each measurement.
Recovery of skin barrier function after insertion of a dissolving microneedle patch intohairless rat skin in vivo. The electrical impedance of untreated skin was measured beforetreatment of each group to serve as an internal negative control. The experimental groupsrepresent: (A) untreated skin (negative control) (B) skin treated with a CMC microneedlepatch inserted for 24 h, (C) skin treated with a CMC microneedle patch inserted for 3 sand then removed and (D) skin treated with a CMC microneedle patch inserted for 3 s andremoved, and then covered with a needle-free CMC patch backing.
All groups were occludedfor the first 24 h and unoccluded for the next 24 h. Data represent the average ±standard deviation, n=6 for each group.Guided by this baseline measurement, a CMC microneedle patch (Group B) wasapplied to the skin for 24 h. Impedance measurements could not be made during this timebecause the patch was in the way. Immediately after removing the patch at 24 h, skinimpedance had dropped to approximately 10 2 kΩcm 2,due to the combined effects of microneedle penetration into the skin and extendedocclusion by the patch. Over time after patch removal, skin impedance increased (ANOVA, p 0.05), indicating complete recovery ofthe skin barrier.As a final comparison, we again pierced the skin transiently with a CMCmicroneedle patch and then occluded the skin with a separate needle-free CMC patch backing(Group D). In this way, the microneedles punctured holes in the skin, but did not dissolvein the skin, and the site remained occluded. Once again, impedance measurements could notbe made during the first 24 h due to interference of the applied patch.
After removal ofthe occlusion, skin impedance of Group D showed recovery that was significantly less thanthe untreated control, Group A (ANOVA, p. Skin reaction to microneedle administrationAs an additional assessment of safety, skin reaction to hGH delivery usingmicroneedles was determined by measuring erythema and edema on a 4-pointscale andimaging the skin. There was no skin edema observed in any of the experimental groups. As shownin, thesubcutaneous injection groups also exhibited no erythema at any time from 24 h afterinjection. CMC and CMC/trehalose microneedle patches showed slight erythema upon patchremoval (Student's t-test, p.
DiscussionThis work presents a detailed study of hGH delivery using a dissolving microneedlepatch designed for simple, painless self-administration that generates no biohazardous sharpwaste. Conventional administration of biopharmaceuticals like hGH requires hypodermicinjection, which requires patients to either visit the clinic for each injection or receivetraining to self-administer the medication. Perhaps the greatest potential impact of thedissolving microneedle patch described in this study is the ability to replace hypodermicneedles and thereby empower patients to self-administer biopharmaceuticals by reducingpatient apprehension, removing the need for expert administration and minimizing safetyconcerns.This study addressed delivery of hGH because this biopharmaceutical isconventionally administered to children by hypodermic injection multiple times per week forup to years.
Currently, there are approximately 20,000 patients per year in the UnitedStates taking hGH and approximately 4,000 per year are estimated as new candidates for hGHtreatment. Thus,hGH delivery serves not only as a useful model compound to study delivery ofbiopharmaceuticals using dissolving microneedles, but also represents a drug that couldsignificantly benefit from administration using a microneedle patch.A dissolving microneedle patch can provide a number of safety advantages comparedto hypodermic needles.
First, dissolving microneedle patches generate no biohazardous sharpwaste, because the microneedles dissolve in the skin and disappear. Indeed, they have the potential to generate no medicalwaste at all, because the patch backing is also made of water-soluble polymer that caneasily be eliminated by dissolving in water in the toilet or sink. This safety featureremoves the risk of accidental needle-stick injury or intentional reuse of needles, which iscommon in some developing countries and is responsible for close to one million deaths peryear due to transmission of hepatitis B, HIV and other infectiousdiseases.Safe use in the skin was addressed in this study and suggested that dissolvingmicroneedles can be safely inserted into the skin. Only slight, transient erythema withoutedema was observed after microneedle treatment. However, it may be of concern that the skinbarrier was slow to fully recover and associated with highly localized inflammatoryresponses, which we hypothesize, were due to slow clearance of residual microneedle matrixmaterial in the skin. Changing microneedle formulation to comprise materials more readilycleared from the skin could increase the rate of local skin recovery.A critical element of dissolving microneedle design was the encapsulation of hGHwithout damaging its functional integrity. By designing a moderate-temperature, water-basedfabrication process, we found that hGH retained full activity after microneedle fabricationas assessed both in vitro and in vivo.
After extended storage for months, some loss ofactivity was measured. This stability assessment was carried out using microneedlesformulated only with CMC. The addition of trehalose to the formulation, which was done inthis study primarily to expedite dissolution time, may also further increase hGH stability,because trehalose is known to stabilize biomolecules during storage.
For further reference, the suggestedshelf life for commercial lyophilized hGH is 2 years at 2-8°C and the reconstitutedsolution can be stored for approximately 2 weeks at 2-8°C. Mechanistic studies have shown thathGH is sensitive to degradation by oxidation, for example, storage of lyophilized hGH in thepresence of approximately 0.75% oxygen at room temperature for 6 months resulted in12% decomposition by oxidation. In this study, hGH encapsulated in dissolving microneedlepatches was exposed to air containing 21% oxygen at 23°C and lost15% and 40% activity after 3 or 15 months, respectively. It thereforeappears that hGH encapsulated in air-dried microneedles affords similar stability asconventional lyophilized hGH and that storage in inert gas or under vacuum that reducesoxidative damage could enable long-term stability of hGH in dissolving microneedlepatches.Bioavailability of hGH administered by a dissolving microneedle patch wasapproximately 30% lower than subcutaneously injected hGH, which was shown to becaused by incomplete insertion and dissolution of the microneedles in the skin. Morecomplete microneedle dissolution could be achieved by improving the microneedle formulationby further increasing matrix material water solubility, by modifying microneedle geometryand insertion method to increase the depth of penetration into skin, and by localizing drugmore toward the microneedle tip.In conclusion, this study addresses the limitations of hypodermic injection ofbiopharmaceuticals by presenting a dissolving microneedle patch designed for safe and simpledelivery of hGH for self-administration by patients. Microneedle patches were shown toencapsulate hGH without loss of functional activity and to exhibit good stability afterstorage up to 15 months in air at room temperature.
Our data in rats demonstrated71% bioavailability of hGH, where the remaining hGH was mostly accounted for in thepatch due to incomplete dissolution of microneedles. Treatment with dissolving microneedleswas well tolerated by the skin with only slight and transient erythema. The skin barrierstayed open for up to 2 days after patch removal, which was probably due to residualmicroneedle matrix in skin. Overall, this study demonstrates the feasibility of a dissolvingmicroneedle patch for delivery of hGH and other biomolecules.
Fabrication of dissolving microneedle patchDissolving microneedles were fabricated using molding techniques and a modifiedsolvent-casting method described previously. First,microneedle master structures were fabricated by using UV photolithography processes withSU-8 photoresist (SU-8 2025, Microchem, Newton, MA), as shown in.
Then, an inverse mold was created by casting masterstructures in polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI).To serve as the CMC microneedle matrix material, ultra-low viscositycarboxymethylcellulose (CMC, Cat No. 360384, Aldrich, Milwaukee, WI) was dissolved indeionized water and then dehydrated to form a viscous hydrogel, the concentration of whichwas approximately 25% w/v. To make CMC/trehalose microneedles, CMC andD-(+)-trehalose dihydrate (Cat No. T9531, Sigma, St. Louis, MO) were mixed at aratio of 1:1 and similarly concentrated to approximately 25% w/v. Then,recombinant human growth hormone (hGH, Genotropin, Pfizer, Groton, CT) was added by handmixing to form a homogeneous mixture in the concentrated hydrogel containing human growthhormone and matrix material at a mass ratio of 1:9.To encapsulate hGH within both the microneedle shaft and the patch backing, thehydrogel containing hGH was cast onto the mold and dried under centrifugation, asdescribed previously and shown in.
To encapsulate hGH within themicroneedle shaft only, the hydrogel containing hGH was cast into the mold cavities onlyand then pure hydrogel without hGH was applied onto the mold surface to form the hGH-freepatch backing, and then the system was dried under centrifugation. For the skin resealingstudy, patches without microneedles were made by applying only pure hydrogel without hGHonto a mold without microneedle cavities.
Animal modelWild-type male hairless rats (10-11 weeks old, 280-340 g, CD Hairless Rat,Charles River, Wilmington, MA) were used for in vivo hGH delivery experiments, withapproval by the Institutional Animal Care and Use Committee of Georgia Tech. They wereanesthetized with isoflurane (ISOTHESIA, Butler Animal Health Supply, Dublin, OH) duringinsertion of microneedles, measurement of skin impedance for the skin resealing study anddrawing blood for the pharmacokinetics study. For the skin reaction study, skin treatedwith microneedles was observed for up to 1 week and then excised after euthanasia, fixedusing formalin, embedded with paraffin, and sectioned for histology.
Functional activity of hGHhGH functional activity was determined by calibrated measurement ofhGH-stimulated growth of Nb2 rat lymphoma cells , ,purchased from Sigma-Aldrich. This study was composed of three phases; (1) cell growth inthe growth-promoting culture medium to determine the normal growth rate of Nb2 cells for 3days, (2) transfer of Nb2 cells to the stationary culture medium to suppress cell growthfor 1 day and (3) addition of hGH test solutions to Nb2 cells incubated in the stationaryculture medium to measure cell proliferation induced by hGH for 3 days. All types ofculture medium were prepared by following the methods describedpreviously. Thenumber of viable Nb2 cells at each phase was measured using a cell viability analyzer(Vi-CELL, Beckman Coulter, Miami, FL). Five different hGH test solutions were added to theNb2 cell culture in phase 3; (i) Placebo solution having only CMC reconstituted from amicroneedle patch (negative control), (ii) hGH solution (Genotropin, positive control),(iii) hGH solution mixed with CMC reconstituted from a microneedle patch, (iv) hGH and CMCsolution reconstituted from a CMC microneedle patch encapsulating hGH, and (v) hGH and CMCsolution reconstituted from a CMC microneedle patch encapsulating hGH after 3 monthsstorage at ambient conditions (23±2°C and 38±5% relativehumidity).
For all groups, the mass ratio of hGH to CMC in the whole system is 5:95, whichmeans that 5 wt% hGH was loaded into the CMC microneedle patch. Pharmacokinetics of hGHThe pharmacokinetic study involved five groups of hairless rats, as summarizedin.
Group1 was administered a subcutaneous injection of 169 ± 10 μg hGH in theGenotropin formulation as received from the manufacturer, which served as the positivecontrol. Group 2 was administered a CMC microneedle patch encapsulating 148 ± 5μg hGH. Group 3 was administered a subcutaneous injection of a reconstituted CMCmicroneedle patch encapsulating 162 μg hGH after 15 months storage at ambientconditions (23±2°C and 38±5% relative humidity). Group 4was administered a CMC/trehalose microneedle patch encapsulating 164 ± 14μg hGH. Finally, Group 5 was administered a subcutaneous injection of areconstituted CMC/trehalose microneedle patch encapsulating 167 ± 15 μghGH. Subcutaneous injections were performed using a 27G hypodermic needle (BectonDickinson, Franklin Lakes, NJ) inserted into the center of the back and dissolvingmicroneedle patches were inserted into the lower part of the back by gentle pressing witha thumb, as described previously.
In groups 2 and 4, microneedle patches were covered with a dressing (Tegaderm,3M Health Care, St. Paul, MN), and then the animal's torso was bandaged withself-adherent wrap (Coban, 3M Health Care) and affixed with adhesive tape (Zonas; Johnsonand Johnson, Skillman, NJ). The microneedles were secured in this way for 24 h tofacilitate drug delivery during microneedle dissolution and prevent patch removal ordisturbance by the animals.
In some cases, a rodent e-collar (404 ¼VS, WebsterVeterinary, Sterling, MA) was placed around the animal's neck.At 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after hGH administration, 120 μlof blood was drawn from the saphenous vein in the tail by a minor incision with a surgicalblade and collected in a CAPIJECT tube (T-MG, Terumo Medical, Elkton, MD). The collectedblood was left at room temperature for 2 h and then spun at 1200×g for 10 min(5415 R centrifuge, Eppendorf, Westbury, NY) to isolate serum, which was transferred intoa 0.5 ml conical tube. After storage at -70°C, hGH concentration was determined byenzyme-linked immunosorbent assay (ELISA) and a microplate reader (iMark, Bio-Rad,Hercules, CA) using a kit specific for hGH without cross-reaction with endogenous ratgrowth hormone (ACTIVE DSL-10-1900, Diagnostic Systems Laboratories, Webster, TX). Areasunder the concentration curve (AUC) were computed by the trapezoid method and used tocalculate bioavailability.
Skin impedance measurementsSkin resealing after insertion of CMC microneedle patches was monitored bymeasuring skin electrical impedance by adapting a method from a related study carried outin humans. Anelectrical impedance meter, (Prep-Check EIM-105, General Devices, Ridgefield, NJ) applied30 Hz AC current between a reference electrode (4.5 cm 2) coated with highlyconductive gel (Superior Silver Electrode with PermaGel, Uni-Patch, Wabasha, MN) and anadjacent, disposable Ag/AgCl dry electrode (0.8 cm 2) (T3404, ThoughtTechnology, Stens Corporation, San Rafael, CA) at the site of microneedle insertion. Theskin impedance was measured in 4 groups.
Group A received no treatment (negative control).Group B was administered a CMC microneedle patch left in place for 24 h. Group C wasadministered a CMC microneedle patch that was inserted and then removed within 3 s, i.e.,before significant microneedle dissolution could occur. Finally, Group D was administereda CMC microneedle patch that was inserted and removed within 3 s, and then covered with aCMC patch backing (i.e., without microneedles attached) for 24 h. In all cases, the skinwas occluded using bandages as described above for the first 24 h and then unoccluded forthe second 24 h. In Groups A and C, skin impedance was monitored for the full 48 hexperiment, where the measurement electrode was left in contact with the skin for thefirst 24 h under occlusion and only briefly contacted the skin for each measurement duringthe second 24 h without occlusion. In Groups B and D, skin impedance was measured onlyduring the second 24 h period, because the presence of the microneedle patch backingprevented impedance measurements during the first 24 h. Skin reactionTo study skin reactions after treatment with hGH dissolving microneedles, theinsertion site was imaged by digital photography (FZ50, Panasonic, Tokyo, Japan) at 24,27, 30, 36, and 48 h and then daily for 5 additional days to assess erythema and edemausing a 0-4 point scale for each.
After euthanasia, treated skin sites were biopsied andprepared for histology by tissue fixing with 10% neutral buffered formalin,paraffin embedding, slicing into 1 μm thick sections with a rotary microtome, andstaining with hematoxylin and eosin for viewing by brightfield microscopy (E600, Nikon,Tokyo, Japan). Representative images of hairless rat skin in vivoduring recovery after hGH administration. Each group is the same group as in Figure S5.The skin was occluded for the first 24 h and unoccluded afterwards.Figure S2.
Skin erythema rating after treatment withmicroneedles. Erythema scale: 0 = normal skin, 1 = slight, 2 =mild, 3 = moderate, and 4 = severe erythema. Each group is the same asdefined in Figure S5. Skin erythema was not rated during the first 24 hours, because itwas occluded and could not be seen. No edema was seen in any groups. Data pointsrepresent the average ± standard deviation for n ≥ 4 for up to 72hours.Figure S3. Representative histological images of skin biopsiedfrom hairless rats in vivo.
Each group is the same as described in Figure S5. At 24 h,Groups 2 and 4 show the sites of microneedle insertion (arrows) and appear to undergohighly localized inflammatory responses (see Figure S4 for magnified views).Figure S4. Representative histological images under highmagnification of skin biopsied from hairless rats in vivo.
(A) Normal skin (negativecontrol). (B) Subcutaneously injected skin (Group 1).
Skin at sites of microneedleinsertion 24 h after treatment with a (C) CMC microneedle patch (Group 2) and (D)CMC/trehalose microneedle patch (Group 4). Note the presence of cells at the sites ofinsertion in (C) and (D) (arrows), which appear to be associated with inflammation.Figure S5. Summary of hGH formulation and administration. Group1: subcutaneous injection of hGH, Group 2: CMC microneedle patch encapsulating hGH,Group 3: subcutaneous injection of reconstituted CMC microneedle patch encapsulating hGHafter 15 months storage at ambient condition, Group 4: CMC/trehalose microneedle patchencapsulating hGH, and Group 5: subcutaneous injection of reconstituted CMC/trehalosemicroneedle patch encapsulating hGH. Seungkeun Choi for the help with the microneedle master fabrication and Prof.Ajay K.
Banga for helpful demonstration of the design of in vivo administration of the patchto the animal skin. This work was carried out at the Georgia Tech Center for Drug Design,Development and Delivery and Institute for Bioengineering and Biosciences and was supportedin part by the National Institutes of Health. Serves as a consultant and is aninventor on patents licensed to companies developing microneedle-based products. Contributor InformationDr.
Jeong Woo Lee, School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332.Dr. Seong-O Choi, School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332.Prof. Felner, Division of Pediatric Endocrinology, Hughes Spalding Children's Hospital, Emory University School of Medicine, Atlanta, GA 30322.Prof.
Prausnitz, School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332.
Hello,I have Visual C installed, and I am able to create programs and compile them using the IDE, but I cannot find the actual file of the compiler anywhere on my computer. I know it is cl.exe is the compiler, but doing a search on my hard drive yields no results.I looked in the Microsoft.NET folder but it is not one of the applications there (along with csc.exe, etc.).
I really would like to compile from my command line by adding cl.exe to my system path, but I can't do that until I actually find the location of cl.exe.Thank you very much for any help. Do you know if there is any way to run cl.exe without running the batch file every time? I'm assuming I would have to add some location to my system path, but I'm not sure which path would enable me to run cl.exe without any errors(from experience I know C:Program Files (x86)Microsoft Visual Studio 10.0VCbin doesn'twork. I don't want to add some huge directory to my path though (maybe I can but my intuition is telling me too large a directory in the path is bad?).Yes, adding stuff to PATH and is bad, and vcvars32.bat is created to avoid the enviroment pollution.
If you don't like it - another bat file can help.Put it in your project directory and click to open a command window where you can compile: pushd '%dp0'call your VCINSTALLPATHVCvcvarsall.batcmd /k- pa. Why not simply use: Start, All Programs, Microsoft Visual Studio 2010, Visual Studio Tools, and select Visual Studio Command Prompt (2010)?
You can issue a cl.exe command directly from there.I have always had problems with that, probably just because I am a novice at using it. This makes it so that the cl.exe is accessible, but now the program I have to compile isn't.
So I have to type 'cl.exe (some long path)', which is just the same problem asI had before but now in a new format: before I would have to type '(some long path ending in /cl.exe) program.cc'. It would have been better to start off your own question.But if you don't have a VC directory, then does this mean you didn't install the Desktop development with C workload and at least the VC 2017 version 15.9 v14.6 latest v141 tools (and maybe the Windows 10 SDK), or the Universal WindowsPlatform development workload with the C Universal Windows Platform tools.If you did remember to install these in the Visual Studio installer, then the fastest and easiest thing to try is to just repair install Visual Studio.This is a signature. Any samples given are not meant to have error checking or show best practices. They are meant to just illustrate a point. I may also give inefficient code or introduce some problems to discourage copy/paste coding.
This is becausethe major point of my posts is to aid in the learning process.