Fukui 2012 Liposome Encapsulate

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Liposome-Encapsulated Hemoglobin Accelerates SkinWound Healing in Mice

*Tsuyoshi Fukui, †Akira T. Kawaguchi, ‡Susumu Takekoshi, *Muneo Miyasaka,and *Rica Tanaka

Departments of *Plastic Surgery, †Cell Transplantation and Regenerative Medicine, and ‡Pathology, Tokai UniversitySchool of Medicine, Kanagawa, Japan

Abstract: Effects of liposome-encapsulated hemoglobinwith high O2 affinity (m-LEH, P50O2 = 17 mm Hg) on skinwound healing in mice were examined. Two full-thicknessdorsal wounds 6 mm in diameter encompassed by siliconestents were created in Balb/c mice. Two days later (day 2),the animals randomly received intravenous m-LEH (2 mL/kg, n = 12), homologous blood transfusion (red blood cell[RBC], n = 11), or saline (n = 12). The same treatment wasrepeated 4 days after wounding (day 4), and the sizes of theskin defects and ulcers were monitored on days 0, 2, 4, and7, when all animals were euthanized for morphologicalstudies. While the size of the skin defect in relation to thestent ring remained the same in all groups, the size of theulcer compared with the skin defect (or silicone stent)became significantly reduced on days 4 and 7 in mice

treated with m-LEH (46 � 10% of pretreatment size, P <0.01) compared with mice treated with RBC transfusion(73 � 6%) or saline (76 � 7%). m-LEH treatment sig-nificantly accelerated granulation, increased epithelialthickness, suppressed early granulocyte infiltration, andincreased Ki67 expression in accordance with the ulcer sizereduction, while there was no difference in surface bloodflow or CD31 expression among the groups. The resultssuggest that m-LEH (2 mL/kg) may accelerate skin woundhealing in Balb/c mice via mechanism(s) involving reducedinflammation and increased metabolism, but not byimproved hemodynamics or endothelial regeneration.Key Words: Artificial oxygen carrier—Skin ulcer—Ischemia—Hypoxia-inducible factor 1a—Hypoxia—Ki67—CD31—Liposome-encapsulated hemoglobin.

Skin wound healing is important not only for accel-erating recovery but also for preventing propagationof inflammation or likelihood of infection. As topicalmalperfusion with resultant ischemia and/or hypoxia(1) has been considered the leading cause of persis-tent ulcer rather than anemia (2) or malnutrition (3),studies have sought effective treatments, includingtopical decompression (4) and hyperbaric oxygentherapy (5). However, topical decompression is notalways possible, and hyperbaric oxygen therapy issometimes difficult to apply, depending on the clinicalsituation and anatomy behind the disease (5).

Therefore, most patients are left with indirect mea-sures to support wound healing, such as transfusionto correct anemia (2) and parenteral alimentation toimprove nutritional status (3).We have been studyingthe action of liposome-encapsulated hemoglobin(LEH) as an artificial O2 carrier (6–8), buffer, or anti-oxidant at reperfusion. Its characteristics includenanometer size, comparable O2-carrying capacity tored blood cells (RBCs), and adjustable O2 affinityallowing targeted O2 delivery (6–8). Unlike the exist-ing cell-free hemoglobin-based O2 carriers (9), hemo-globin is separated by the liposome capsule, similar instructure to RBCs, protecting it against nitric oxidescavenging and resultant adverse effects (9). Becauseof its size (250 nm), which is much smaller thanRBCs, LEH was considered to freely perfuse collat-erals and capillaries and protect the brain fromischemic and/or reperfusion damage in a rodent (6,8)as well as in a primate model (7). Moreover, LEH hasbeen modified to have a higher O2 affinity (h-LEH,

doi:10.1111/j.1525-1594.2011.01371.x

Received March 2011; revised June 2011.Address correspondence and reprint requests to Dr. Akira T.

Kawaguchi, Tokai University School of Medicine, Shimokasuya143, Isehara, Kanagawa 259-1193, Japan. E-mail: [email protected]

Artificial Organs36(2):161–169, Wiley Periodicals, Inc.© 2012, Copyright the AuthorsArtificial Organs © 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

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P50O2 = 10 mm Hg) to transport more O2 than RBCsunder hypoxic conditions (10). Based on thesecharacteristics, we evaluated the hypothesis thatLEH with moderately high O2 affinity (m-LEH,P50O2 = 17 mm Hg) may improve microcirculationand supply O2 to promote aerobic metabolism in skinwounds and thereby accelerate healing in a murinemodel of skin defect, a simulation of skin ulcerhealing.

MATERIALS AND METHODS

LEHThe characteristics of LEH (Terumo Co. Ltd.,

Tokyo, Japan) have been reported (6–8,10). Briefly, itis a liposome capsule measuring 250 nm in meandiameter, containing hemoglobin eluted fromdonated human RBCs outdated for transfusion. Theliposome capsule is coated with polyethylene glycolto reduce aggregation and capture by the reticuloen-dothelial system, allowing its prolongation of circula-tion half-life to 13 h in the rodent (6–8) and to 70 h innonhuman primates (7,11). Inositol hexaphosphatewas included as an allosteric effecter for 2,3-diphosphoglycerate so as to increase O2 affinity to ahigher level (P50O2 = 17 mm Hg, m-LEH) than that ofrodent RBCs (P50O2 = 30 mm Hg) in the currentstudy. LEH is suspended in saline to a hemoglobinconcentration of 6 g/dL or 20% of volume (LEHcrit).LEH is precipitated between plasma and RBC by acentrifuge at 50 000 ¥ g for 2 h. A sibling mousedonated homologous blood, which was collectedin a syringe containing citrate-phosphate-dextrosesolution. The blood was then centrifuged to separateRBCs, which were diluted with saline and washed atleast three times to 20% hematocrit to serve as acontrol solution containing a comparable amount ofhemoglobin to LEH.

Animals and skin woundingAll experiments were approved by the institu-

tional review board of Tokai University School ofMedicine. Animals received humane care asrequested in National Institutes of Health publica-tions (1985) amended in 2005. Male Balb/c micewere purchased from CLEA Co. Ltd. (Yokohama,Japan) and used at 8 weeks of age (20 � 1 g). Micewere anesthetized with 2% sevoflurane, and trunkhair was removed by skin hair remover on the daybefore skin defect creation (Fig. 1A). On day 0, skinwounds were created using a circular skin tome(6 mm diameter) in the bilateral back of the anes-thetized mice (Fig. 1B). A donut-shaped siliconeskin holder (Fuji Systems, Yokohama, Japan) was

fixed around each skin defect, first by adhesivesand then by eight 6-0 monofilament sutures(Fig. 1B) to prevent skin contraction. These skindefects were photographed and covered with anocclusive skin protector to prevent contamination.Finally, a plastic corset was placed around the trunkof the body to keep animals from reaching thewounds themselves.

Drug administrationTwo days later (day 2), animals were randomly

assigned to three groups so that each group with the

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FIG. 1. Experimental protocol. Experimental protocol (A) isillustrated. A mouse received two dorsal skin lesions (B) sur-rounded by a silicone skin holder (a), which was fixed in placefirmly by an adhesive and eight 6-0 monofilament sutures. Thewound (b) was evaluated by photograph to measure the areainside the ring (area 1), skin defect (area 2), and ulcer (area 3)(c).

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Artif Organs, Vol. 36, No. 2, 2012

same size of ulcer received the initial infusion;2 mL/kg of body weight of m-LEH (n = 12), transfu-sion of homologous washed RBCs at the sameamount of hemoglobin (n = 11), or saline (n = 12) wasintravenously infused from the tail vein under 2%sevoflurane anesthesia (Fig. 1A). Each solution wasdiluted five times with saline (approximately 0.2 mLper animal) for volume accuracy at administration.After administration, animals were placed back incages in room air, with access to food and water adlibitum until 2 days later for the second medication(day 4) and 5 days later for the last observation andsacrifice (day 7, Fig. 1A).

Laser Doppler flow measurementFour additional mice (n = 4) in each group, treated

in the same way, were followed for surface blood flowand euthanized for histological observation on day 4,2 days after initial treatment (Fig. 1A). To evaluatechanges in surface blood flow in response to theadministration of solutions, surface perfusion wasmonitored in normal skin (area 1, Fig. 1B), edge ofthe ulcer (area 2), and center of the ulcer (area 3)before and after solution administration on day 2 byLaser Perfusion Imager (moorFLPI, Moor Instru-ments, Devon, UK). This was repeated 2 days later,on day 4, when the animals were then sacrificed forhistological studies (Fig. 1A).

Skin lesionsThe area of each skin lesion was measured from

photos taken on day 2 as ring (inside the ring, area 1,Fig. 1B), skin defect (faintly dotted area 2), and ulcer(darkly dotted area 3). Then, the area ratios, skindefect/ring (area 2/area 1), ulcer/ring (area 3/area 1),and ulcer/skin defect (area 3/area 2) were determinedfor the right and left wounds, which served as control(100%) for the ratios of the later observations ondays 4 and 7.

Morphological studiesAfter all the animals were photographed on days 4

(n = 12) and 7 (n = 35), they were sacrificed underdeep anesthesia, and the back skin lesions (two ineach animal, Fig. 1B) were removed in full depth formorphological studies. Each lesion was divided downthe middle; one half was placed in 4% paraformalde-hyde for Ki67 staining and methanol for CD31 stain-ing, and the other half was frozen in liquid nitrogenfor later studies. The ulcer specimen was prepared sothat the wound could be observed in full length alongthe ulcer diameter. The specimens were fixed andstained with hematoxylin–eosin (H&E) for histo-pathological observations, to determine the degree of

neutrophil infiltration, titrated as none (scored 0),mild (scored 1), moderate (scored 2), and severe(scored 3) by two independent observers blinded tothe study protocol. Granulation development andepithelial thickness were also measured in the sameway for each edge of the ulcer (Figs. 1B and 4A).

Immunohistochemical studiesImmunohistochemical staining for hypoxia-

inducible factor 1a (HIF-1a), CD31 (12), and Ki67(13) was carried out. CD31- and Ki67-positive cellnumbers and whole cell numbers as background werecounted by two independent observers. Five fieldswere randomly selected and counted using digitalimage analyzing software (Image J1.37v) developedat the National Institutes of Health, Bethesda, MD.

StatisticsThe two skin lesions in each animal were consid-

ered independently. Data were presented asmean � standard deviation. The variables from eachlesion were averaged for each group, and comparedamong groups by Kruskal–Wallis analysis. A P valueof less than 0.05 was considered significant.

RESULTS

Laser Doppler flow measurementThe surface blood flow determined by Laser

Doppler flow meter (Fig. 2) was greater in the orderof the center, edge of the ulcer, and skin surroundingthe defect. There were no significant or consistentchanges between before and after administration ofsolution (day 2) and 2 days later (day 4). Among theanimal groups, the m-LEH-treated mice had a ten-dency toward reduced flow on day 4 compared withthe animals treated with RBC or saline.

Ulcer sizeThe process of skin wound healing was followed

(Fig. 3A); only lesions properly created according tothe study design were included (day 0). Two dayslater, animals were randomly assigned to threegroups so that each group had the same ulcer size,and they received the initial infusion (day 2). Twodays later (day 4), some differences in macroscopicappearance of the ulcers were noted, and theanimals received the second infusion of the samesolution. Three days thereafter (day 7), the ulcerswere dry and largely covered by epithelium in them-LEH-treated mice, whereas ulcers were still wetin the other treatment groups (Fig. 3A). There was ahighly significant correlation between the left andright lesions in ulcer/skin defect (area 3/area 2) on

ARTIFICIAL O2 CARRIER ACCELERATES WOUND HEALING IN MICE 163


day 4 (r = 0.827, P < 0.05) as well as on day 7(Fig. 3B, P < 0.01). When bilateral lesions were inde-pendently evaluated in the time sequence (Fig. 3C),skin defect/ring (area 2/area 1) was reduced withoutdifference among the treatment groups, and ulcer/ring (area 3/area 1) and ulcer/skin defect (area3/area 2) were significantly more reduced inm-LEH-treated mice than in mice receiving RBC orsaline, with no difference between the latter twogroups on day 4 or on day 7.

Granulation and epitheliumGranulation formation and epithelial thickness

were determined in each lesion (Fig. 4A) and aver-aged for each treatment group. Granulation wasbetter developed in mice treated with m-LEH(617 � 142 mm, P < 0.05) compared with mice treatedwith RBC (486 � 125 mm) or saline (446 � 116 mm).Also, the epithelium was thicker in mice treatedwith m-LEH (114 � 27 mm, P < 0.05) than in micereceiving RBC transfusion (70 � 24 mm) or saline(74 � 17 mm) on day 7.

Neutrophils, Ki67-positive cells, andCD31-positive cells

HIF-1a was not clearly stained in any animal onday 4 or day 7. Granulocyte infiltration and Ki67- andCD31-positive cells were counted in each lesion (Fig.5A), and averaged for the treatment groups at 2 days(day 4) after the initial treatment and 3 days (day 7)after the second administration (Fig. 5B). Althoughgranulocyte infiltration was significantly suppressedin the m-LEH-treated mice (1.4 � 0.5, P < 0.05) com-pared with mice treated with RBC (2.3 � 0.7) orsaline (2.8 � 0.5) on day 4, the infiltration subsided in

the other treatment groups by day 7, and no signifi-cant difference was observed among the groups.While CD31-positive cells showed no significant dif-ference among the m-LEH (114 � 29), RBC(104 � 33), and saline (95 � 9) groups, Ki67-positivecells were significantly more numerous in them-LEH-treated mice (115 � 17, P < 0.05) than inmice treated with RBC (96 � 14) or saline (95 � 9)on day 4, and also on day 7 (m-LEH-treated mice,104 � 14, P < 0.05; RBC, 86 � 10; saline, 84 � 8).

DISCUSSION

LEH has been examined on an experimentalbasis and was found to be protective in animalmodels of cerebral ischemia (6–8,10), cochlearischemia/reperfusion (14), skeletal muscle ischemia/reperfusion (15), gastrointestinal wound healing (inpreparation), and enhancing tumor radiotherapy(16). Improved microcirculation, increased oxygendelivery, and persistent aerobic energy metabolismform the rationale for these applications of LEH(17). In particular, LEH with high O2 affinity is con-sidered to be able to, and has actually beenobserved to, efficiently deliver O2 to tissues underischemia (10) or deranged perfusion after gastricsurgery (in preparation). Based on these experimen-tal results, the current study was performed toexamine our hypothesis that LEH may acceleratewound healing of the skin, where O2 metabolismmay be different from other organs or tissues(6–8,14–16).

Since surface blood flow as determined by LaserDoppler flow meter (Fig. 2) showed no differencesamong the treatment groups on days 2 and 4, hemo-dynamic effects were likely not responsible for the

FIG. 2. Changes in surface blood flow. Infour additional mice (n = 4) in each grouptreated in the same way, surface bloodperfusion was monitored in normal skin(area 1), edge of the ulcer (area 2), andcenter of the ulcer (area 3) before andafter the administration of the solution onday 2 and again on day 4 by Laser Perfu-sion Imager (moorFLPI, Moor Instruments,Devon, UK).

Edge (Area 2)Skin (Area 1) Center (Area 3)

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significant differences among the treatment groups.As normal skin structure would not be able todevelop within the observed 7-day period, there wasno difference in the size of the skin defect in terms ofring area among the treatment groups. Each mousehad two dorsal skin lesions; the degree of healing inthe right and left lesions was similar (Fig. 3B), sug-gesting that wound healing was largely dependent onthe individual animal than the construction or main-tenance of each lesion.

Hypoxia immediately after skin wounding isknown to increase the production of angiogenic

factors such as vascular endothelial growth factor,platelet-derived growth factor receptor, and trans-forming growth factor-b and their dose-dependenteffect on angiogenesis (18). In the current study,m-LEH was administered 2 days after wounding,when these immediate hypoxia and/or angiopro-liferative signals had already been generated andangiogenesis triggered in each animal, accounting forlittle or no expression of HIF-1a in each animal andno significant difference in CD31 expression amongtreatment groups. While the first 2-day observationwithout treatment allowed us to equally subject the

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FIG. 3. Macroscopic changes in ulcers. Macroscopic sequential observation was carried out in all animals (A). There was a highlysignificant correlation between the left and right lesions in ulcer/skin defect (area 3/area 2) on day 4 (r = 0.827, P < 0.05), as well as onday 7 (B). When bilateral lesions were independently evaluated in the time sequence (C), skin defect/ring (area 2/area 1) decreasedwithout difference among the treatment groups, ulcer/ring (area 3/area 1), and ulcer/skin defect (area 3/area 2) were significantly morereduced in m-LEH-treated mice than in mice receiving RBC or saline (*P < 0.05), with no difference between the latter two groups.



wounds to the initial ischemic phase and toevenly randomize the animals regarding the woundin terms of size, coverage, and absence of infection,later oxygenation by m-LEH on days 2 and 4 mighthave caused the significant reduction in ulcerousareas. In fact, these macroscopic and functionalobservations were in accordance with the histologicalfindings; both granulation and epithelium were sig-nificantly better developed in the m-LEH-treatedmice compared with the other two groups, whichshowed no difference between them.

In order to examine the mechanism(s) involved inthe benefits of m-LEH treatment, neutrophil infil-tration to the lesions was counted. Consistent with

the macroscopic observations, the infiltration ofneutrophils was suppressed in m-LEH-treated mice2 days (day 4) after the initial treatment as com-pared with the other treatment groups. The differ-ence was no longer significant 3 days following thesecond treatment (day 7), highlighting the possibil-ity that m-LEH treatment suppresses inflammatoryreaction early after skin wounding. Such early sup-pression of inflammatory response appears to be acommon phenomenon after LEH administration,as it was also observed following gastric surgery(in preparation). While HIF-1a was not clearlystained in any of the animals on day 4 or day 7,Ki67 expression was increased only in the

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FIG. 4. Microscopic observations. Granulation formation and epithelial thickness were determined in each lesion (A) and averaged for thetreatment group (B). Both granulation and epithelial thickness were significantly more accelerated in the m-LEH-treated mice than in micetreated with RBC or saline (*P < 0.05), with no difference between the latter two groups.

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FIG. 5. Immunohistochemical observations. Granulocyte infiltration (H&E, 300¥), CD31-positive cells (200¥), and Ki67-positive cells(200¥, arrows) were counted in each lesion (A), and averaged for each treatment group (B) at 2 days (day 4) after the initial treatmentand at 3 days (day 7) after the second administration. Although granulocyte infiltration was significantly suppressed only in them-LEH-treated mice on day 4 (*P < 0.05), the extent became less in the other treatment groups on day 7, when there was no differenceamong the groups. While the number of CD31-positive cells showed no significant difference among the groups on day 7, Ki67-positivecells (arrows) were significantly more numerous in the m-LEH-treated mice than in the other mice both on days 4 and 7 (*P < 0.05).



m-LEH-treated animals on these days, suggestingthat protein synthesis was accelerated comparedwith the other treatments. Such elevated synthesiswas apparently not related to the regeneration ofblood vessels, as CD31 expression did not differamong the groups. Thus, LEH administration pro-motes skin wound healing not by angiogenesis, butby increasing the fibroblast proliferation and col-lagen synthesis. Although the 5-day follow-up withtwo m-LEH 2 mL/kg administrations in the currentstudy may not be long enough for vascular regen-eration, LEH might have promoted wound healingby increasing fibroblast proliferation, as confirmedby the increased Ki67 expression and collagen for-mation observed in the accelerated granulation andepithelial development.

Transfusion is a common therapeutic option forreversing anemia, replacing blood loss, improving O2

delivery, and accelerating postoperative recovery(2). Although most artificial O2 carriers have beendeveloped for this purpose, so far, no single producthas been accepted as an RBC substitute for clinicaluse (9). LEH as blood substitute has been reportedby Ikegawa and colleagues (19), who demonstratedthat LEH with low O2 affinity (P50 = 40 mm Hg)may be inferior to RBCs in maintaining systemicmetabolism and peripheral perfusion in rabbitsundergoing extended exchange hemodilution up to86%. As the current LEH has high O2 affinity(P50 = 17 mm Hg) as compared with RBC (P50 =30 mm Hg in mice), it is not likely to unload oxygenprematurely to induce vasoconstriction or tissuehypoperfusion, but rather deliver O2 to hypoxictissue efficiently (17). In contrast, RBC trans-fusion failed to improve skin wound healing in thecurrent study, similar to mice receiving saline. Ashematocrit in all animals was considered to benormal, it is conceivable that the additional RBCtransfusion might not add any benefit as alsosuggested by Mandai et al. (2), who revealed thathemoglobin over 10 g/dL was enough in woundhealing. Similar protective effects of LEH havebeen demonstrated in cerebral ischemia andreperfusion models in rats (6,8) as well as in non-human primates (7), another model of local perfu-sion derangement. As RBC transfusion failed to bebeneficial in either of these cases, the nanometersize of LEH may in fact provide advantages overRBC transfusion in animal models of cerebralischemia and reperfusion as well as wound healing.Thus, the presence of hemoglobin-containing nano-particles in plasma, but not the presence of hemo-globin in the form of RBCs, may be important forwound healing.

CONCLUSION

The current results support our hypothesis thatm-LEH may improve aerobic metabolism andaccelerate wound healing in skin ulcer, which is notoffered by homologous transfusion, nor by anequivalent amount of hemoglobin in the form ofRBCs. The mechanism is not clear from this study,but it is probably related to a suppressed immediateinflammatory response and later proper oxygen-ation for fibroblast proliferation and collagen syn-thesis in the skin defect, and likely not via directhemodynamic effect or accelerated angiogenesis, apossible mechanism in other therapeuticapproaches. In fact, such treatments might providesynergistic effects. Optimal oxygen affinity, dose, andtiming of LEH administration will need to be clari-fied for their application.

Acknowledgments: This study was supported inpart by: (i) Grant-in-Aid for Scientific Research14370365, 16209037, and 20249072 from the Ministryof Education, Culture, Science and Technology,Tokyo, Japan; (ii) New Energy Development Organi-zation (NEDO), Tokyo, Japan; and (iii) JST, Tokyo,Japan. We would like to thank Chihiro Fujinuma,Naokatsu Ando, Yo Kawaguchi, and HideyukiHanano (Tokai University School of Medicine) fortheir help with the experiments and animal care. Wealso thank the Tokai University Teaching andResearch Support Center, Research DevelopmentDivision for assistance with the experimental setup,preparation, and administration.

Conflict of Interest: All authors, Tsuyoshi Fukui,Akira T. Kawaguchi, Susumu Takekoshi, MuneoMiyasaka, and Rica Tanaka, are clinicians/scientistswho organized and performed this study. At the timethis work was conducted and completed, they allworked at the Tokai University School of Medicine,where all animal experiments took place.Terumo Co.Ltd. developed and supplied the LEH tested in thisstudy. ATK received the research grants specifiedabove, which provided materials and personnel tocarry out the experiments and summarize the resultsin this manuscript. There were no other monetarydependencies or conflicts of interest among theauthors.

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Fukui 2012 Liposome Encapsulate