Sarcoidosis with heart involvement: a rare association of terrible prognosis, a report of two cases Case report Open Access
2015

Asma Kefi
Department of Internal Medicine
Department of Internal Medicine
Military Hospital of Tunis
1008MontfleuryTunisia

Military Hospital of Tunis
1008MontfleuryTunisia

Nadia Ben Abdelhafidh
Department of Internal Medicine
Department of Internal Medicine
Military Hospital of Tunis
1008MontfleuryTunisia

Military Hospital of Tunis
1008MontfleuryTunisia

Sameh Sayhi
Department of Internal Medicine
Department of Internal Medicine
Military Hospital of Tunis
1008MontfleuryTunisia

Military Hospital of Tunis
1008MontfleuryTunisia

Rim Abid
Department of Internal Medicine
Department of Internal Medicine
Military Hospital of Tunis
1008MontfleuryTunisia

Military Hospital of Tunis
1008MontfleuryTunisia

Faida Ajili
Department of Internal Medicine
Department of Internal Medicine
Military Hospital of Tunis
1008MontfleuryTunisia

Military Hospital of Tunis
1008MontfleuryTunisia

Janet Laabidi
Department of Internal Medicine
Department of Internal Medicine
Military Hospital of Tunis
1008MontfleuryTunisia

Military Hospital of Tunis
1008MontfleuryTunisia

Salah Othmani
Department of Internal Medicine
Department of Internal Medicine
Military Hospital of Tunis
1008MontfleuryTunisia

Military Hospital of Tunis
1008MontfleuryTunisia

Asma Kefi
Sarcoidosis with heart involvement: a rare association of terrible prognosis, a report of two cases Case report Open Access

Pan African Medical Journal -ISSN
21201510.11604/pamj.2015.21.243.6124Received: 13/01/2015 -Accepted: 07/04/2015 -Page number not for citation purposes 1 & Corresponding author: Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Page number not for citation purposes 2Cardiac sarcoidosisSarcoidosisVentricular arrhythmias
Sarcoidosis is a multisystemic disorder of unknown etiology which is characterized by the formation of non-caseating granulomas in involved tissues. Cardiac involvement is one of the least common manifestations and it can occur at any point of time during the course of sarcoidosis. Here we present the case of 2 patients with known sarcoidosis who develop cardiac abnormalities in the absence of known primary cardiac cause. In our report, we would like to draw attention to the importance of considering heart involvement in any case with systemic sarcoidosis especially in young age.

Introduction

Sarcoidosis, an enigmatic multisystemic disease, is characterized by the formation of granulomas in many tissues mainly the lungs, the lymphoreticular system, the eyes, and the skin, with cardiac involvement being a rare entity [1]. Many reports showed that the quoted incidence of heart involvement in sarcoidosis in their review was around 2%, so that it was one of the least common manifestations [1]. The clinical sequelae of cardiac sarcoidosis vary from asymptomatic conduction abnormalities to heart failure, fatal ventricular arrhythmias, and sudden death. Foregoing data, early diagnosis and prompt treatment are essential since they can improve prognosis [2] by reducing the chances of sudden death.

Patient and observation Case 1

A 40-year-old Tunisian woman, with a history of diabetes mellitus in diet and Lofgren's syndrome (an acute presentation consisting of polyarthralgia, erythema nodosum, bilateral intrathoracic lymphadenopathies) diagnosed in February 2013, came to our department, in February 2014, for exploration of exertional dyspnea of one month evolution and progressively worsening without fever, chest pain, or syncope. The patient denied any past history of hypertension, coronary artery, or thyroid disease. She did not smoke or consume alcohol. On physical examination, she was conscious, cooperative, and afebrile. She was hemodynamically stable (blood pressure (BP) 130/70 mmHg and had a regular pulse of 88 beats /minute.). She had good color and was slightly polypneic with no peripheral cyanosis (peripheral oxygen saturation 97%). She presented no palpable adenopathy or signs of jugular distension. Cardiac auscultation revealed rhythmic sounds with no murmur or friction rub.There were no signs of peripheral edema or deep vein thrombosis. Pulmonary, abdominal, and neurological examination revealed no abnormalities. The admission electrocardiogram (ECG) was normal. On investigation, blood biochemistry, liver, renal function, thyroid function and, hemogram showed no abnormalities except lymphopenia. Her angiotensin converting enzyme (ACE) level was 132 units (normal range < 68units). The immunological markers were negative. A contrastenhanced computed tomography (CT) chest scan showed almost complete regression of mediastinal lymphadenopathies without pleuroparenchymal lesion. Respiratory functional tests and bronchoscopy with transbronchial lung biopsy were normal. The transthoracic echocardiography with Doppler showed abnormal left ventricular relaxation and systolic dysfunction of the left ventricle with ventricular ejection fraction (EF) of 40%. In the light of these symptoms and echocardiographic abnormalities, for which other possible causes have been excluded, the diagnosis of sarcoidosis with heart involvement was suspected. Thus, more specialized cardiac evaluation was needed. 24 hour holter monitoring showed recurrent episodes of 2000 monomorphic ventricular extrasystoles. Cardiac magnetic resonance imaging (MRI) with gadolinium contrast showed evidence of delayed hyper enhancement in both the basal and inferolateral left ventricular regions, suspicious for fibrogranulomatous tissue of sarcoidosis ( Figure 1). The left ventricular ejection fraction was 40%. Laboratory tests confirmed negative serial markers of myocardial necrosis. Technetium99m sestamibi scintigraphy myocardial perfusion study showed decreased uptake in the inferolateral and apical ventricular myocardium, which was reversible during stress (reverse distribution) ( Figure 2). Coronary angiography and let ventriculography showed angiographically normal coronary arteries. Considering all these clinical and investigatioanal findings in a patient with a history of Lofgren's syndrome and in the absence of primary heart disease, the diagnosis of cardiac sarcoidosis was made. The patient started with a dose of 1mg/kg/day of corticosteroids in addition to supportive care. This dose was tapered gradually to a maintenance level of 15 mg per day over 5 months. To this day, evolution was favorable.

Case 2

A 48-year-old Tunisian woman, presented to our consultations with a history of intermittent palpitations and exertional dyspnea of 4 months duration, progressively worsening for 2 weeks before, without any history of chest pain or syncope. She had a past history of systemic sarcoidosis diagnosed in April 2009 on the basis of the following compatible clinical and radiologic findings: systemic symptoms (such as fatigue, anorexia, and weight loss), parotid enlargement, negative tuberculin skin test, lymphopenia, hypercalciuria, increased serum ACE level, intrathoracic lymphadenopathies which were hilar ( Figure 3), bilateral symmetrical, and associated with right paratracheal window lymph node involvement. Pulmonary functions tests demonstrated decreased volumes and CO diffusing capacity. Lymphocytosis in broncho-alveolar lavage was observed and a CD4+/CD8+ lymphocyte ratio was greater than 3.5. Lip biopsy of accessory salivary glands was compatible with grade 1 according to Chisholm's classification. After ruling out the other granulomatous diseases such as tuberculosis, lymphoma, crohn's disease, Wegner granulomatosis..., the diagnosis of systemic sarcoisosis was ascertained. A short-course corticosteroid therapy with antimalarial drug were initiated with a favorable evolution both clinically and radiogically.The patient was presented in November 2011 with a history of intermittent palpitations and exertionaldyspnea of 4 months duration, progressively worsening for 2 weeks before. She denied any history of hypertension, diabetes mellitus, coronary artery disease and thyroid disease. She did not smoke, consume alcohol, or illicit drugs. On physical examination, she was conscious, cooperative, and afebrile. She had a BP of 120/70 mmHg and a pulse of 160 beats/minute. There were no signs of cardiac failure; the rest of the physical examination did not reveal any abnormal findings. She was admitted to the cardiac intensive care unit. The admission ECG showed a ventricular tachycardia at 160 cycles/minute. In the light of these typical symptoms and electrocardiographic alterations in a patient having systematic sarcoidosis, the diagnosis of sarcoidosis with cardiac involvement was suspected. The echocardiography showed a FE of 50%, bright shadows consisting with infiltration in the basal and inferoseptal ventricular myocardium, and moderate mitral regurgitation. Coronary angiography revealed normal coronary arteries. The cardiac electrophysiology study showed a monomorphic ventricular tachycardia induced by isoproterenol. Laboratory tests showed no relevant alterations in myocardial necrosis biomarkers, blood cell count, renal and liver function, or electrolytes. Given the absence of primary heart disease that would explain the cardiac abnormalities and especially the previous diagnosis of systemic sarcoidosis, the final diagnosis was sarcoidosis with cardiac involvement. The patient started with a dose of 1 mg/kg/day of corticosteroid, in addition to supportive care. Over 8 months, the steroids were tapered to a dose of 10 mg/day. Anti arrhythmic drug therapy (Amiodarone) and β blocker were initiated. Given the recurrent ventricular tachycardia with amiodarone, we proceeded to the implantation of an automated implantable cardioverter-defibrillator (ICD). Evolution was favorable.

Discussion

Sarcoidosis is a multisystemic disorder of unknown etiology which is characterized by the formation of non-caseating granulomas in involved tissues. To this day, it remains something of enigma, the search for an etiologic agent is ongoing [2]. Sarcoidosis can be widespread or limited to involvement of only a single system at a time, it most commonly involves the lungs, the lymph nodes of the thorax and the eye, despite the fact that virtually no organ systems are immune to infiltration by sarcoid granulomas. Cardiac involvement is one of the least common manifestations and it can occur at any point of time during the course of sarcoidosis. Vishal S et al reported that the incidence of clinical heart involvement was as approximately 5%, wheras at autopsy, the incidence was considerably higher (20 to 25%) [2].

Making a diagnosis of cardiac sarcoidosis remains one of the main challenges in the management of the disease: in fact, if cardiac manifestations occur in patients with multisystemic sarcoidosis, the diagnosis, although circumstancial, is strongly suspected, which is in keeping with our 2 cases. However, when cardiac dysfunction is the only manifestation of sarcoidosis, the diagnosis is frequently not entertained. Clinical manifestations of cardiac sarcoidosis, ranging from asymptomatic conduction abnormalities to fatal ventricular arrhythmia or sudden death, are dependent on both the profusion and location of granulomas. Granulomas can involve the left ventricular free wall, basal ventricular septum, right ventricle, papillary muscle, right atrium, and left atrium [3]. Main relevant signs include complete heart block, ventricular tachycardia, supra ventricular arrhythmias (atrial flutter, fibrillation and paroxysmal atrial tachycardia), congestive heart failure, and sudden death. Other clinical manifestations may involve chest pain, electrocardiographic changes, and pericardial abnormalities (pericardial effusion, constrictive pericarditis, tamponade) [2]. Despite the fact that endomyocardial biopsy is theoretically the most confident mean to assert the diagnosis of cardiac sarcoidosis, it is an invasive procedure which lacks sensitivity: cardiac involvement tends to be patchy and granulomas are more likely to be located in the left ventricle and basal ventricular septum than in the right ventricle where endomyocardial biopsies are usually performed [4]. Thus, serial ECG during evolution survey, echocardiography, 24-h Holter monitoring of ECG, MRI with gadolinium contrast, technetium99m-sestamibi scintigraphy myocardial perfusion study, and coronary angiography can be helpful tools for diagnosis. However, all these complementary exams lack specificity for the disease [5]. Thus, in 1993, guidelines for the diagnosis of cardiac sarcoidosis have been published by the Japanese Ministry of Health and Welfare [6]. The 2 cases presented fulfill the diagnostic criteria set out in these guidelines ( Table 1): cardiac abnormalities in the absence of primary heart disease with a previous diagnosis of intrathoracic sarcoidosis in the first case and Löfgren's syndrome in the second case. The clinical response to the treatment prescribed also supports our diagnosis suspicion.

Considering the increased risk of sudden death, cardiac sarcoidosis is an indication for early treatment. Steroids should be started as soon as possible: many reports showed that, in early or middle stage disease, steroid therapy may be protective or therapeutic but may not be as effective in the late stages [7]. In both our cases, treatment was started at the middle stage of the disease and a favorable response was noted.The recommended starting dose seems to be 60 to 80 mg a day of prednisone, it can be tapered over 6 months to a dose of 10 mg a day [2]. Antiarrhythmic drugs and β blockers are also usually used in the management of cardiac sarcoidosis. To our knowledge, there have been no prospective studies evaluating the use of these medications in patients having cardiac sarcoidosis. Amiodarone can be responsible for the occurrence of pulmonary fibrosis. These drug-induced pulmonary changes, which are indistinguishable radiographically from pulmonary sarcoidosis, may threaten the patient's respiratory status. Consequently, we must carefully and constantly assess the potential benefits and risks of prescribing such medications. Regarding the treatment with ICD, it seems necessary in patients with refractory ventricular tachyarrhythmia who are at risk of sudden death in addition to antiarrhythmic drugs.

Some authors recommended ICD implantation in any patient with sarcoidosis and non-sustained ventricular tachyarrhythmia because of the high rate of recurrence of ventricular tachycardia despite medical treatment [8,9]. As for cardiac transplantation, it is rare and remains the only possibility for younger patients with severe and stage irreversible cardiac failure or resistant VT [10]. The prognosis of cardiac sarcoidosis remains obscure: early necropsy series concluded that survival in most patients with symptomatic cardiac sarcoidosis was limited to about 2 years [3], while better outcomes were noted in other series where 5-year survival rate was 40-60%. Whether this improvement in prognosis was due to early disease recognition or a milder form of cardiac sarcoidosis versus early institution of medication remains to be determined. With progress in prevention and treatment of ventricular arrhythmias, the primary cause of death in cardiac sarcoidosis has changed from sudden death to congestive heart failure.

Conclusion

Cardiac sarcoidosis should always be suspected in any patient who is younger than expected presenting with cardiac symptoms without evidence of primary heart disease particularly if he has already been diagnosed with sarcoidosis. To date, early diagnosis of cardiac sarcoidosis remains a challenge because clinical manifestations are non specific and the diagnostic modalities lack sensitivity and specificity in spite of recent advances. A negative endomyocardial biopsy should in no way be regarded as evidence of absence of cardiac sarcoidosis, especially if diagnostic suspicion is strong. Cardiac sarcoidosis is on occasion a medical emergency that may lead to sudden death due to the rhythm disturbances or progressive heart failure from a dilated cardiomyopathy; therefore, in addition to corticosteroid treatment, early consideration for implantation of an ICD should be a priority. Table and figures   Table 1: the Japanese Ministry of Health and Welfare 1993 guidelines for the diagnosis of cardiac sarcoidosis

Figure 1 :
1cardiac magnetic resonance imaging (MRI) with gadolinium contrast: evidence of delayed hyper enhancement (arrow) in both the basal and inferolateral left ventricular regions

Figure 2 :Figure 1 :
21decreased uptake in the inferolateral and apical ventricular myocardium, which was reversible during stress (reverse distribution) shown on Technetium99m sestamibiscintigraphy myocardial perfusion studyFigure 3: high-resolution computed tomography scan of the chest showing peribronchial and bilateral hilar lymphadenopathy (arrows) cardiac magnetic resonance imaging (MRI) with gadolinium contrast: evidence of delayed hyper enhancement (arrow) in both the basal and inferolateral left ventricular regions

Figure 2 :
2decreased uptake in the inferolateral and apical ventricular myocardium, which was reversible during stress (reverse distribution) shown on Technetium99m sestamibiscintigraphy myocardial perfusion study

Figure 3 :
3high-resolution computed tomography scan of the chest showing peribronchial and bilateral hilar lymphadenopathy (arrows)
Competing interestsThe authors declare no competing interests.Authors' contributionsAuthors read and approved the final version of the manuscript. Cardiac sarcoid is confirmed then endomyocardial biopsy demonstrates epithelioid granulomas without caseating necrosis.2: clinical diagnosis groupin patients with a histologic diagnosis of extracardiac sarcoidosis, cardiac sarcoidosis is suspected when: "a" and at least one of criteria "b" to "e" is present, and other etiologies such as hypertension and coronary artery disease have been excluded.a Bundle branch block, heart block of any degree, left-axis deviation, ventricular tachycardia, premature ventricular contraction, or pathological Q or ST-T change on resting or ambulatory electrocardiogram. bAbnormal wall motion, regional wall thinning, or dilation of the left ventricle.c Perfusion defect by thallium.201 or technetium-99m myocardial scintigraphy, or abnormal accumulation by gallium 67.d Abnormal intracardiac pressure, low cardiac output, or abnormal wall motion or depressed ejection fraction of the left ventricle on cardiac catheterization.e Interstitial fibrosis or more than moderate cellular infiltration over moderate grade on endomyocardial biopsy specimen, even if the findings are non-specific.
Clinical characteristics of patients in a case control study of sarcoidosis. R P Baughman, A S Teirstein, M A Judson, M D Rossman, Am J Respir Crit Care Med. 16410PubMed | Google ScholarBaughman RP, Teirstein AS, Judson MA, Rossman MD et al. Clinical characteristics of patients in a case control study of sarcoidosis. Am J Respir Crit Care Med. 2001; 164(10):1885- 1889. PubMed | Google Scholar

Cardiac sarcoidosis: a comprehensive review. V Sekhri, S Sanal, L Delorenzo, W Aronow, G Maguire, Arch Med Sci. 74PubMed | Google ScholarSekhri V, Sanal S, DeLorenzo L, Aronow W, Maguire G. Cardiac sarcoidosis: a comprehensive review. Arch Med Sci. 2011; 7(4): 546-554. PubMed | Google Scholar

Sarcoidosis of the heart; A clinicopathologic study of 35 necropsy patients (group I) and review of 78 previously described necropsy patients (group II). W C Roberts, H A Mcallister, V J Ferrans, Am J Med. 631PubMed | Google ScholarRoberts WC, McAllister HA, Ferrans VJ. Sarcoidosis of the heart; A clinicopathologic study of 35 necropsy patients (group I) and review of 78 previously described necropsy patients (group II). Am J Med. 1977; 63(1): 86-108. PubMed | Google Scholar

Histologic diagnostic rate of cardiac sarcoidosis: evaluation of endomyocardial biopsies. A Uemura, S Morimoto, S Hiramitsu, Y Kato, T Ito, H Hishida, Am Heart J. 1382PubMed | Google ScholarUemura A, Morimoto S, Hiramitsu S, Kato Y, Ito T, Hishida H. Histologic diagnostic rate of cardiac sarcoidosis: evaluation of endomyocardial biopsies. Am Heart J. 1999; 138(2):299-302. PubMed | Google Scholar

Diagnosis of cardiac sarcoidosis. M Silverman, J Oshinski, R Pettigrew, Chest. 1102PubMed | Google ScholarSilverman M, Oshinski J, Pettigrew R. Diagnosis of cardiac sarcoidosis. Chest. 1996; 110(2):562-565. PubMed | Google Scholar

Guideline for diagnosis of cardiac sarcoidosis: study report on diffuse pulmonary diseases from the Japanese Ministry of Health and Welfare. Tokyo: Japanese Ministry of Health and Welfare. H Hiraga, K Yuwai, M Hiroe, 2324PubMed | Google ScholarHiraga H, Yuwai K, Hiroe M et al. Guideline for diagnosis of cardiac sarcoidosis: study report on diffuse pulmonary diseases from the Japanese Ministry of Health and Welfare. Tokyo: Japanese Ministry of Health and Welfare. 1993; 23: 24. PubMed | Google Scholar

Prevention of left ventricular remodeling by long-term corticosteroid therapy in patients with cardiac sarcoidosis. C Z Chiu, S Nakatani, G Zhang, Am J Cardiol. 951PubMed | Google ScholarChiu CZ, Nakatani S, Zhang G, et al. Prevention of left ventricular remodeling by long-term corticosteroid therapy in patients with cardiac sarcoidosis. Am J Cardiol. 2005; 95(1):143-146. PubMed | Google Scholar

Sustained ventricular tachycardia associated with sarcoidosis: assessment of the underlying cardiac anatomy and the prospective utility of programmed ventricular stimulation, drug therapy and an implantable antitachycardia device. S L Winters, M Cohen, S Greenberg, J Am Coll Cardiol. 184PubMed | Google ScholarWinters SL, Cohen M, Greenberg S et al. Sustained ventricular tachycardia associated with sarcoidosis: assessment of the underlying cardiac anatomy and the prospective utility of programmed ventricular stimulation, drug therapy and an implantable antitachycardia device. J Am Coll Cardiol. 1991; 18(4): 937-943. PubMed | Google Scholar

The automated implantable cardiac defibrillator; Prophylaxis in cardiac sarcoidosis. H L Paz, D J Mccormick, S P Kutalek, Chest. 1065PubMed | Google ScholarPaz HL, McCormick DJ, Kutalek SP et al. The automated implantable cardiac defibrillator; Prophylaxis in cardiac sarcoidosis. Chest. 1994; 106(5): 1603-7. PubMed | Google Scholar

Cardiac sarcoidosis: response to steroids and transplantation. H A Valantine, H D Tazelaar, J Macoviak, J Heart Transplant. 64PubMed | Google ScholarValantine HA, Tazelaar HD, Macoviak J et al. Cardiac sarcoidosis: response to steroids and transplantation. J Heart Transplant. 1987; 6(4): 244-50. PubMed | Google Scholar


A cost-effective handheld breast scanner for use in low-resource environments: a validation study

Robyn B Broach
Rula Geha
Brian S Englander
Lucy Delacruz
Holly Thrash
Ari D Brooks
A cost-effective handheld breast scanner for use in low-resource environments: a validation study
10.1186/s12957-016-1022-2R E S E A R C H Open Access
Background: With the incidence of breast cancer rising worldwide, we are evaluating the iBreastExam (iBE) (UE LifeSciences Inc.), a handheld breast scanning device that can be utilized by community health workers to screen for breast abnormalities. The purpose of this study is to determine the sensitivity of the iBE in a population undergoing diagnostic breast imaging.Methods: Adult patients presenting to a breast imaging center for a diagnostic workup were eligible. Patients underwent an iBE exam performed by a trained ultrasound technician followed by their indicated imaging. Demographic, imaging, and biopsy data were recorded.Results: Seventy-eight iBE exams were completed, 77 females and one male with a mean age of 42 (21-79). All patients were evaluated by ultrasound, 52 had diagnostic mammography and 39 had biopsies. Imaging and/or biopsy confirmed a mass (fibroadenoma, cyst, papilloma, myofibroblastoma, fat necrosis, DCIS, or cancer) in 60 patients. Twelve patients had a cancer diagnosed. In total, 342 quadrants were scanned, 77 quadrants had lesions confirmed on imaging, and iBE correctly identified 66 lesions for a sensitivity of 86 % and specificity of 89 %.Conclusions: This validation study demonstrated excellent sensitivity of iBE for the identification of clinically significant lesions in patients presenting for diagnostic imaging. Trial registration: A Cost-Effective Handheld Breast Scanner for Use in Low Resource Environments: A Validation Study: NCT02814292.

Background

The incidence of breast cancer is rising rapidly worldwide. Since 2008, the incidence of breast cancer has increased by more than 20 % worldwide [1]. Globally, breast cancer now represents one in four of all cancers among women. Early detection improves the survival rate, makes treatment less costly, and lowers the overall burden of the disease. The iBreastExam (iBE) device was developed as a pre-screening tool that could identify women in need of further breast imaging without requiring extensive breast screening infrastructure. This inexpensive handheld device uses piezoelectric palpation to enhance the clinical breast exam (CBE) for detection of breast masses that require further investigation.

The iBE device was built on the principle of the piezoelectric finger (PEF) detector. After their initial development, the PEF was proven in bench-top work on breast phantoms and subsequently in excised human tumors with excellent detection ability and size prediction [2]. A pilot in vivo clinical trial was then completed using a very basic array of four PEFs with excellent detection of breast lesions in women undergoing clinical evaluation [3]. Subsequently, the iBE device was developed as a 16 finger array with a rapid wireless mobile processor algorithm and durable battery powered handpiece. The device was developed to be operated by a technician or health care worker and does not require a radiologist for interpretation. This prospective study was designed specifically to validate the ability of the iBE device to detect breast abnormalities worthy of further diagnostic imaging.

Methods

Study design

Approval was obtained from the University of Pennsylvania Institutional Review Board (IRB, protocol # 819121) and the Abramson Cancer Center Clinical Trials Scientific Review Committee (CTSRMC, UPCC # 33113) for this study. All patients signed informed consent prior to participation.

Eighty-nine patients with suspected breast lesions were consented and examined with the iBE device from August 2014 through January 2015. Patients participated for one clinic visit during which the iBE device was used to examine the areas of concern as well as uninvolved areas of the breast. Eleven patients were excluded from the analysis due to missing the iBE data. Missing iBE data was a result of incomplete data transfer from the iBE. If the wireless communication of the device was incomplete, repeat scans were not attempted due to time constraints of the high volume at the breast imaging center. Results from the iBE did not influence or change the medical care given to the patients.

Women and men 18 years of age or older with symptomatic breast masses or women with asymptomatic breast masses discovered by palpation or imaging were included. Patients under 18 years of age were excluded.

The current pathway of breast lesion screening and detection at this institution begins with a screening mammogram in asymptomatic women. In women with a negative screening mammogram, the pathway is complete and a regular interval follow-up is recommended. This tends to be the case even in women with dense breast tissue. In women with an abnormal screening mammogram or palpable masses from clinical breast exam, a diagnostic mammogram is done in women over 40 and an ultrasound in all women. Typically, if a woman is under 35 with a palpable mass, mammography is skipped and ultrasound is the diagnostic test of choice. If the additional imaging reveals something suspicious, a biopsy may be recommended for confirmation. Since the inclusion criteria required the participating patients to have a mass detected by palpation or imaging, the majority of the patients were recommended for further imaging. All follow-up diagnostic information was extracted from the electronic medical record and recorded. The type and number of diagnostic techniques were determined by the radiologist and/or the surgical oncologist based on the results of the previous exams, not including the iBE.

Classification

Initially in this study, a positive or a negative finding was defined only by the Breast Imaging-Reporting and Data System (BIRADS) classification assigned by the radiologist interpreting the results of the diagnostic technique(s). BIRADS 1 and 2 defined a negative detection while BIRADS 0, 3, 4, 5, and 6 defined a positive detection or clinically relevant finding. However, it was discovered that many of the radiology reports classified as BIRADS 1 or 2 reported a measurable finding such as a cyst. Since the iBE was designed to be a pre-screening tool with the intent to identify all lesions, not just cancerous lesions, the positive/negative classification for this study was revised to include the presence or absence of clinically relevant detectable lesions as follows: negative findings included negative study, fibrocystic changes, benign calcification, and gynecomastia while positive findings included fibroadenoma, cyst, myofibroblastoma, fat necrosis, papilloma, ductal carcinoma in situ (DCIS), or cancer. Thus, when analyzing the iBE results by independent quadrants, each iBE quadrant was compared to the correlating quadrant of a mammography, ultrasound, biopsy, or MRI. A positive finding was defined as the presence of a measurable finding in that quadrant independent of BIRADS classification.

Device

The iBreastExam™ or iBE™ ( Fig. 1a, b) consists of a handheld compression probe containing a 4 × 4 array of piezoelectric tactile pressure sensors, a custom built electronic board and a tablet. The iBE communicates wirelessly with a mobile device to display and store the findings in real time. Compression data are recorded as a unique file at the end of every scan to the mobile device which is synced to an encrypted database via Dropbox where a copy is saved. No identifiable data are collected or stored by the iBE software.

The iBE evaluations were performed by an ultrasound technologist who was trained to use the iBE by the manufacturer and the principal investigator of the study. Training involved 1 h of education about the functions of the device, practice on a breast phantom, and then 10 observed iBE studies with the principal investigator. The results of the iBE were displayed on the touch screen in a pressure map fashion (Fig. 1c, d). Green indicates normal breast tissue while red indicates a lesion was detected meaning the iBE is suggesting further testing to characterize the lesion, as the iBE does not differentiate the type of lesion it detects. The patient lies in the supine position while the test was done. The device was calibrated on the breast in an uninvolved area; it can be recalibrated multiple times. The iBE records the data and collects into 4 × 4 array map of the breast, and each individual square represents the 4 × 4 array of the PEFS. This map was divided into sectors demarcated by three consecutive hours of a clock in order to directly compare to the clock positioning assigned by mammography or ultrasonography.

Results

The mean age of the patients was 42 years old (age range 21 to 79 years). The racial distribution of the patient population was 51 % Caucasian, 35 % African American, 3 % Asian, 5 % Hispanic, and 6 % Unknown. One male participated in the study. All 78 patients underwent ultrasonography while only 52 underwent mammography and 39 underwent biopsies (50 %).

The iBE results were evaluated by definitive quadrants. The definitive quadrants break down the superimposed clock into four equal sectors (e.g., 12-3, 3-6, 6-9, [9][10][11][12]. The quadrants were defined when the iBE identified a positive finding; the positive finding became the center of a quadrant, meaning if a positive finding was identified at 5:00, the first quadrant would be 3:30-6:30 and the remaining quadrants defined as follows 6:30-9:30, 9:30-12:30, and 12:30-3:30.

Through imaging, 77 of the quadrants were determined as a positive detection and 265 as negative findings with an overall total of 342 quadrants evaluated by the iBE ( Table 1). Out of the 77 positive quadrants, 66 were identified correctly (true positive) by the iBE resulting in a Sn = 85.7 %, and 237 out of 265 quadrants were correctly characterized normal (true negative) resulting in Sp = 89.4 %. Also, illustrated in Table 1 are the positive detection rate, negative detection rate, and accuracy, positive predictive value (PPV) = 70.2 %, negative predictive value (NPV) = 95.6 %, and accuracy (Acc) = 88.6 %, respectively. Table 2 demonstrates the distribution of BIRADS categories according to the patient's final report of the evaluation of the entire breast. BIRADS (1 and 2) were assigned to 30 patients, and BIRADS (3, 4, 5, and 6) were assigned to 48 patients. A total of 5 masses went undetected by the iBE, all of which were under 1 cm in size. Of the false negatives, 3 false negatives were classified as BIRADS 3, noncancerous findings while the remaining 2 were cancerous and classified as BIRADS 4 until biopsies were completed. Two of the 3 noncancerous false negatives were fibroadenomas, and the remaining 1 was stromal calcification. One cancerous false negative was retroareolar 0.5cm ductal carcinoma in situ, and the second was 0.7-cm mucinous carcinoma located at 8:30, 6 cm from the nipple.

Malignancy

Of the 78 patients, 12 patients were found to have a malignancy. Ten of the 12 cancerous diagnoses were detected by the iBE (Sn = 83 %, Sp = 74.5 %) while 2 were undetected ( Table 3). The size of malignancies detected ranged from of 0.9 to 3.8 cm with a mean size of 1.91 cm ± 0.9. The two undetected by the iBE mean size was 0.6 ± 0.1 cm. One of the undetected malignancies was ductal carcinoma in situ; the other was mucinous carcinoma. Out of the 10 malignant masses detected by the iBE, 6 were invasive ductal carcinoma, 2 were invasive mammary carcinoma and 1 was ductal carcinoma in situ. Two of the detected masses were located in the retroareolar region. Diagnostic mammograms were performed on 11 of the 12 cancers diagnosed, while one patient had a diagnostic ultrasound only ( Table 4). Ten of the 11 cancers were detected by mammogram (Sn = 91 %, Sp = 51 %) and one undetected, a 1-cm size invasive ductal carcinoma. The iBE detected the cancer the mammography missed; conversely, mammography identified the cancers the iBE missed.

Tables 3 and 4 display the accuracy of malignancy detection of the iBE evaluation and mammography. Table 4 shows the diagnostic mammogram with a total of 32 positive findings. Positive findings for mammograms were defined by the radiologist assigned BIRADS 0, 4, 5, and 6, and negative evaluations were defined as BIRADS 1, 2, and 3. BIRADS 3 is added to the negative evaluation, because BIRADS 3 is considered nonmalignant category. All but one of the BIRADS 0 was eventually determined to be a BIRADS 4 by ultrasound or biopsy. The positive predictive value, negative predictive value, and the accuracy of the mammogram to detect cancer were determined to be 33.3, 95.5, and 59.6 %, respectively. The positive predictive value, negative predictive value, and the accuracy of the iBE to detect cancer were determined to be 10.6, 99, and 74.9 %, respectively.

Discussion

The iBE demonstrated acceptable sensitivity and specificity in this cohort of patients requiring diagnostic imaging for palpable lesions or abnormal screening mammograms. This study was designed to validate the iBE's ability to detect lesions in patients with a positive breast exam or imaging.

Mammography and ultrasonography demonstrate sensitivity rates of 85-88 % [4][5][6][7] and 93-97 % [7-10] for a general screening and diagnostic population, while the iBE in this population of known detectable findings demonstrated a comparable sensitivity of 85.7 %. The performance of this device meets the threshold of a pre-screening device when compared to the current pre-screening tool, CBE, which has a sensitivity of 50-60 % [11,12].

The iBE detected all but two of the malignancies, both of which were less than a 1 cm in size. This threshold is an important one for a pre-screening device in lowresource settings. The goal of early detection is to identify malignancies where intervention will improve survival, possibly with reduced need for expensive and morbid adjuvant therapy. Identifying cancers in stages I and II before they become stages III or IV definitely fulfills this criterion in a low-resource environment [13][14][15][16]. The iBE is designed to be a pre-screener in order to help identify masses that need to be investigated further; therefore, this amplified sensitivity could be valued to detect potentially significant underlying masses.

Directly comparing the sensitivity of cancer detection demonstrates that both the iBE (83 %) and mammography (91 %) are reliable tools to identify patients with cancer (Tables 3 and 4). While the sensitivity of cancer detection is lower, the specificity of the iBE (74 %) surpasses that of mammography (51 %) indicating that the iBE is superior to mammography at not falsely identifying patients with cancer suggesting that the iBE as a pre-screening tool will be less likely to cause unnecessary stress and anxiety.

Reexamining the iBE results to reflect a patient outcome instead of a single breast or a portion of the breast, we determined that 61 of the 78 patients required a mammogram or an ultrasound for further evaluation of the screening results. Of those 61 patients, 56 (91.8 %) were properly recommended by the iBE for a mammogram or ultrasound; however, 5 (8.2 %) were missed by the iBE that needed a mammogram or ultrasound for further information. Based on the imaging results, 17 patients did not require a mammogram or ultrasound, 4 (23.5 %) of them were identified correctly by the iBE while 13 (76.5 %) were not.

All 78 patients were eligible to participate in this study due to positive imaging, clinical breast exam, or a selfbreast exam identifying a suspicious area directing them down the diagnostic pathway, resulting in a 50 % biopsy rate for this study population. This incidental high  biopsy rate was beneficial because the biopsy results confirmed the identity/classification of the suspicious masses detected by the iBE or imaging. We demonstrated the iBE's ability to screen similar to mammography in this cohort. The iBE screenings in this study did not lead to an increase rate of biopsies as the iBE results did not influence the patient's diagnostic pathway. The high biopsy rate in this study was a result of examining a population on the diagnostic testing pathway. The iBE is a sensitive tool that is ready for validation in a large screening cohort. This screening study is already underway, and we hope to have a reliable specificity value from that trial before deploying this device in lowresource settings around the world. Because the device is portable, radiation free, and painless and does not require a radiologist, the iBE is an excellent pre-screening device. When used to select patients for further diagnostic workup with mammogram, ultrasound, and/or biopsy, the iBE has the potential to bring access to breast cancer screening and diagnosis to women who currently have little or no access to screening mammography worldwide.

We see the iBE as a potential powerful screening tool for use in the third world countries with limited resources where mammography and clinical breast exam by a trained physician is not readily available. Positive findings by the iBE could then allow for patients to be referred to larger medical centers where ultrasound and mammography may be available and, more importantly, where surgical excision could be performed. Training lay people on the use of the device could potentially provide screening to rural and underserved areas of undeveloped nations where women historically have had no access to breast cancer screening. The iBE device is currently being piloted in a rural area in India where due to limited economic resources, women have essentially no access to mammography and present with late-stage breast cancers. As described in the literature, late-stage breast cancers have poorer prognosis than early stage breast cancers [16]. Due to the significantly lower cost of the iBE compared to conventional mammography, we see this device as a very useful adjunct to breast cancer detection worldwide in underdeveloped, third world nations with limited medical economic resources and a need for improved breast cancer screening. Indeed, a group in Bangalore, India, has recently reported on the use of the iBE in a population of women presenting for annual health exams [17]. Abnormal iBE studies were followed by directed ultrasound or mammography as confirmatory studies. In that population, a sensitivity of 84 % and specificity of 94 % for the detection of image detected breast lesions.

Conclusions

This validation study demonstrated excellent sensitivity of iBE for the identification of clinically significant lesions in patients presenting for diagnostic imaging. The iBE demonstrates significant potential as a low-cost screening tool in low-resource environments.

Fig. 1 a
1iBE device and the device in use. b The 4 × 4 array of PEFS. c Data pressure map illustration of normal or negative results. d Data pressure map illustration of abnormal or positive results

Abbreviations
Acc: Accuracy; BIRADS: Breast Imaging-Reporting and Data System; CBE: Clinical breast exam; CTSRMC: Clinical Trials Scientific Review Committee; DCIS: Ductal carcinoma in situ; iBE: iBreastExam; IRB: Institutional Review Board; NPV: Negative predictive value; PEF: Piezoelectric finger; PPV: Positive predictive value

Table 1
1iBE accuracy measures by definitive quadrantsTotal definitive
quadrants N = 342

Mass
positive

Mass
negative

Measures

Test positive quadrant
66
28
PPV = 70.2 %

Test negative quadrant 11
237
NPV = 95.6 %

Measures
Sn = 85.7 % Sp = 89.4 % Accuracy = 88.6 %

Table 2
2Distribution of BIRADS and number of FN in each categoryBIRADS
Final report
No. of FN

1
3 (4 %)
0

2
27 (35 %)
0

3
26 (33 %)
3

4
13 (16.7 %)
2

5
1 (1.3 %)
0

6
8 (10 %)
0

Total
78
5

Table 3
3iBE accuracy for malignancy detectionNo cancer
Cancer
Total

IBE (+)
84
10
94

IBE (−)
246
2
248

Total
330
12
342

Measures
Sn = 83 %
Sp = 74.5 %

Table 4
4Mammogram accuracy for malignancy detectionNo cancer
Cancer
Total

Mammogram (+)
20
10
32

Mammogram (−)
2 1
1
2 1

Total
41
11
52

Measure
Sn = 91 %
Sp = 51 %

AcknowledgementsMihir Shah and Matthew Campisi of UE Lifesciences, Inc. are acknowledged for their technical and logistical support in this clinical trial.FundingThis study was supported by Pennsylvania CURE Grant # 4100059198.Availability of data and materialsThe datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request.Authors' contributions ADB conceived the study and the design, assisted in the data analysis and interpretation, and helped draft and critically revised the manuscript. RBB's contributions were data acquisition, analysis, and interpretation and drafting the manuscript. BSE performed the analysis and interpretation of the radiologic data. HT was the device supervisor and lead technician of the data acquisition. RG and LD assisted in the data interpretation. All authors above made significant contributions to the content, design, and revision of this manuscript. The results of this study were presented at the American Society Breast Surgeons annual meeting April 2016, Dallas, TX. All authors read and approved the final manuscript.Competing interestsAri D Brooks, MD, is a co-inventor on the original patent of the device held by Drexel University. Holly Thrash is a support research personnel provided by UE Lifesciences Inc.Consent for publication Not applicable.Ethics approval and consent to participate Approval for this clinical trial was obtained from the University of Pennsylvania Institutional Review Board (IRB FWA00004028, protocol # 819121) and the Abramson Cancer Center Clinical Trials Scientific Review Committee (CTSRMC, UPCC # 33113) for this study. Prior to participation, all patients signed informed consent form approved by the entities listed above.• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
Breast cancer statistics. Wcrf International, International WCRF. Breast cancer statistics. 2012. http://www.wcrf.org/int/ cancer-facts-figures/dataspecific-cancers/breast-cancer-statistics.

Probing model tumor interfacial properties using piezoelectric cantilevers. H H Yegingil, W Y Shin, W H Shih, Rev Sci Instrum. 8195104Yegingil HH, Shin WY, Shih WH. Probing model tumor interfacial properties using piezoelectric cantilevers. Rev Sci Instrum. 2010;81:095104.

Breast tumor detection using piezoelectric fingers: first clinical report. X X Xu, C Gifford-Hollingsworth, R Sensenig, W H Shih, A Brooks, J Am Coll Surg. 216Xu XX, Gifford-Hollingsworth C, Sensenig R, Shih WH, Brooks A. Breast tumor detection using piezoelectric fingers: first clinical report. J Am Coll Surg. 2013;216:1168-73.

Screening breast ultrasound: past, present, and future. R F Brem, M J Lenihan, J Lieberman, J Torrente, Am J Roentgenol. 204Brem RF, Lenihan MJ, Lieberman J, Torrente J. Screening breast ultrasound: past, present, and future. Am J Roentgenol. 2015;204:234-40.

for the Breast Cancer Surveillance C. Performance of digital screening mammography among older women in the United States. L M Henderson, E S O'meara, D Braithwaite, T Onega, Cancer. 121Henderson LM, O'Meara ES, Braithwaite D, Onega T, for the Breast Cancer Surveillance C. Performance of digital screening mammography among older women in the United States. Cancer. 2015;121:1379-86.

Diagnostic accuracy of digital screening mammography with and without computer-aided detection. C D Lehman, R D Wellman, D M Buist, JAMA Intern Med. 175Lehman CD, Wellman RD, Buist DM, et al. Diagnostic accuracy of digital screening mammography with and without computer-aided detection. JAMA Intern Med. 2015;175:1828-37.

Role of breast ultrasound and mammography in evaluating patients presenting with focal breast pain in the absence of a palpable lump. R Leddy, A Irshad, E Zerwas, N Mayes, K Armeson, M Abid, A Cluver, A Campbell, S Ackerman, M Lewis, Breast J. 19Leddy R, Irshad A, Zerwas E, Mayes N, Armeson K, Abid M, Cluver A, Campbell A, Ackerman S, Lewis M. Role of breast ultrasound and mammography in evaluating patients presenting with focal breast pain in the absence of a palpable lump. Breast J. 2013;19:582-9.

Ultrasonographic study of the palpable breast tumor is very useful. C Perre, P De Hooge, P Leguit, Ned Tijdschr Geneeskd. 137Perre C, de Hooge P, Leguit P. Ultrasonographic study of the palpable breast tumor is very useful. Ned Tijdschr Geneeskd. 1993;137:2374-7.

. T M Kolb, J Lichy, J H Newhouse, Radiology. 207Kolb TM, Lichy J, Newhouse JH. Radiology. 1998;207:191-9.

Solid breast nodules: use of sonography to distinguish between benign and malignant lesions. A T Stavros, D Thickman, C L Rapp, M A Dennis, S H Parker, G A Sisney, Radiology. 196Stavros AT, Thickman D, Rapp CL, Dennis MA, Parker SH, Sisney GA. Solid breast nodules: use of sonography to distinguish between benign and malignant lesions. Radiology. 1995;196:123-34.

Findings from 752,081 clinical breast examinations reported to a national screening program from 1995 through 1998. J K Bobo, N C Lee, S F Thames, J Natl Cancer Inst. 92Bobo JK, Lee NC, Thames SF. Findings from 752,081 clinical breast examinations reported to a national screening program from 1995 through 1998. J Natl Cancer Inst. 2000;92:971-6.

Does this patient have breast cancer?: The screening clinical breast examination: should it be done? How?. M B Barton, R Harris, S W Fletcher, JAMA. 282Barton MB, Harris R, Fletcher SW. Does this patient have breast cancer?: The screening clinical breast examination: should it be done? How? JAMA. 1999;282:1270-80.

Decreased rates of advanced breast cancer due to mammography screening in The Netherlands. J Fracheboud, S J Otto, Jaam Van Dijck, Mjm Broeders, Alm Verbeek, H J De Koning, Br J Cancer. 91Fracheboud J, Otto SJ, van Dijck JAAM, Broeders MJM, Verbeek ALM, de Koning HJ. Decreased rates of advanced breast cancer due to mammography screening in The Netherlands. Br J Cancer. 2004;91:861-7.

Improvements in survival for women with breast cancer in Scotland between 1987 and 1993: impact of earlier diagnosis and changes in treatment. C S Thomson, D H Brewster, J A Dewar, C J Twelves, Eur J Cancer. 40Thomson CS, Brewster DH, Dewar JA, Twelves CJ. Improvements in survival for women with breast cancer in Scotland between 1987 and 1993: impact of earlier diagnosis and changes in treatment. Eur J Cancer. 2004;40:743-53.

Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Ebctc Group, Lancet. 365Group EBCTC. Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet. 2005;365:1687-717.

Meeting highlights: international expert consensus on the primary therapy of early breast cancer. A Goldhirsch, J H Glick, R D Gelber, A S Coates, B Thürlimann, H-J Senn, Ann Oncol. 16Goldhirsch A, Glick JH, Gelber RD, Coates AS, Thürlimann B, Senn H-J. Meeting highlights: international expert consensus on the primary therapy of early breast cancer 2005. Ann Oncol. 2005;16:1569-83.

Noninvasive and low-cost technique for early detection of clinically relevant breast lesions using a handheld point-of-care medical device (iBreastExam): prospective three-arm triple-blinded comparative study. S P Somashekhar, R Vijay, R Ananthasivan, G Prasanna, 10.1007/s40944-016-0057-1Indian J Gynecol Oncol. 1426Somashekhar SP, Vijay R, Ananthasivan R, Prasanna G. Noninvasive and low-cost technique for early detection of clinically relevant breast lesions using a handheld point-of-care medical device (iBreastExam): prospective three-arm triple-blinded comparative study. Indian J Gynecol Oncol. 2016; 14:26. doi:10.1007/s40944-016-0057-1.


Bovine serum albumin nanoparticles as controlled release carrier for local drug delivery to the inner ear

Zhan Yu
Min Yu
Zhibao Zhang
GeHong
Qingqing Xiong
Bovine serum albumin nanoparticles as controlled release carrier for local drug delivery to the inner ear
N A N O C O M M E N T A R Y Open AccessBovine serum albuminNanoparticleControlled releaseDrug deliveryRound window membraneInner ear
Nanoparticles have attracted increasing attention for local drug delivery to the inner ear recently. Bovine serum albumin (BSA) nanoparticles were prepared by desolvation method followed by glutaraldehyde fixation or heat denaturation. The nanoparticles were spherical in shape with an average diameter of 492 nm. The heat-denatured nanoparticles had good cytocompatibility. The nanoparticles could adhere on and penetrate through the round window membrane of guinea pigs. The nanoparticles were analyzed as drug carriers to investigate the loading capacity and release behaviors. Rhodamine B was used as a model drug in this paper. Rhodamine B-loaded nanoparticles showed a controlled release profile and could be deposited on the osseous spiral lamina. We considered that the bovine serum albumin nanoparticles may have potential applications in the field of local drug delivery in the treatment of inner ear disorders.

Background

Inner ear disorders, including sensorineural hearing loss (SSHL), commonly occur in clinics. The traditional systemic therapies are almost ineffective due to the bloodlabyrinth barrier, which prevents the transport of drugs from the serum. Local drug delivery, especially intratympanic injection, has become popular for two decades because of its efficiency and safety. The round window membrane (RWM) is a semipermeable membrane between the middle and the inner ear, through which particles less than 3 μm in diameter could penetrate.

Local drug delivery to the inner ear by intratympanic injection was first described by Schuknecht in 1956 in the treatment of Ménière's disease [1]. In 2006, Kopke et al. reported a significant hearing improvement of patients with sudden sensorineural hearing loss after methylprednisolone administration locally [2].

Although intratympanic injection is easy to perform in the clinic, the loss of drug through the Eustachian tube becomes the obstacle to treat inner ear disorders efficiently. Thus, hydrogel-and particle-based vehicles (or carriers) have been investigated recently for sustained and prolonged drug supply. In 1998, Balough et al. described that the local injection of a fibrin-based sustained release vehicle impregnated with gentamicin allowed for a prolonged effect without absorption in the untreated ear or blood [3]. Horie et al. reported that drug-loaded polylactic/glycolic acid (PLGA) microparticles were capable of delivering lidocaine into the cochlea in a sustained manner [4]. The PLGA nanoparticles were found to be distributed throughout the inner ear after application on the RWM of chinchilla [5]. Moreover, Tan et al. demonstrated that brain-derived neurotrophic factor encapsulated in nanoporous poly(L-glutamic acid) particles could be released in a sustained manner with maintained biological activity and efficiently rescue primary auditory neurons in the cochlea of guinea pigs with sensorineural hearing loss [6]. Nowadays, nanoparticles have received much more interest for the treatment of inner ear diseases for their drug loading and sustained release capacity.

Various methods such as desolvation, emulsion, template, microfluidic, mechanical stretching, and self-assembly have been reported to prepare protein-based particles or capsules [7][8][9][10][11]. In this study, we demonstrated that bovine serum albumin (BSA) can form nanospheres by desolvation method and can be used for local drug delivery. BSA is a natural protein able to form complexes in various shapes. This protein is biocompatible, biodegradable, nontoxic, and nonimmunogenic. Due to these features, albumin particles are a good system for drug and antigen delivery [11][12][13][14]. To the best of our knowledge, there have been no reports of local delivery of drug-loaded albumin particles into the inner ear. Here, we illustrate a method for creating sphere-shaped BSA nanoparticles (BSA-NPs) with biocompatibility in high yield. A model drug, rhodamine B (RhB), was loaded onto the BSA-NPs for drug loading capacity, release, and in vivo studies. In vivo biodistribution suggested that the RhB released as well as the RhB-loaded BSA-NPs (RhB-BSA-NPs) tended to accumulate and penetrate through the RWM of guinea pigs. Therefore, the BSA-NPs would be prospectively considered as controlled release carriers for local drug delivery in the treatment of inner ear disorders.

Methods

Materials, mice, and cell culture BSA and RhB were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technology Inc. (Shanghai, People's Republic of China). Ultrapure water used in all experiments was produced by Milli-Q synthesis system (Millipore Corp., Billerica, MA, USA). L929 mouse fibroblast cells (obtained from the Cancer Institute of the Chinese Academy of Medical Sciences, People's Republic of China) were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Thermo Scientific Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS) at 37°C with 5% CO2. Guinea pigs weighing 250~300 g were purchased from the Tianjin Experimental Animal Center, People's Republic of China, and had free access to food and water. Animal study protocols were approved and performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals.

Preparation of BSA-NPs and RhB-BSA-NPs

BSA-NPs were prepared by the desolvation method. Briefly described, 100 mg of BSA was dissolved in 1 ml of sodium chloride solution (10 mM). Then, 8.0 ml of ethanol was added dropwise into the BSA solution under magnetic stirring (400 rpm) at room temperature. Subsequently, the as-prepared BSA-NPs were cross-linked with 0.2% glutaraldehyde (GA) for 24 h or denatured at 70°C for 30 min. BSA-NPs (50 mg) were incubated with certain amounts (5, 10, 15, 17.5, and 20 mg) of RhB for 2 h in the preparation of RhB-BSA-NPs. The particles were centrifuged and washed with ultrapure water.

Characterization of the BSA-NPs

The morphological characteristics were determined by transmission electron microscopy (TEM, JEOL, JEM-100CXII, Akishima-shi, Japan), scanning electron microscopy (SEM, ZEISS SUPRA 55VP, Oberkochen, Germany), and confocal laser scanning microscopy (CLSM, FV-1000, Olympus Corporation, Shinjuku-ku, Japan). For TEM, a drop of diluted suspension of BSA-NPs was placed on the copper grid and the air-dried specimen was observed. For SEM, a drop of diluted suspension was deposited on a silicon wafer. The air-dried sample was coated with gold and observed. RhB-BSA-NPs were observed by CLSM at an excitation wavelength of 555 nm and an emission wavelength of 580 nm.

The BSA-NPs were dispersed in ultrapure water at a concentration of 0.1 mg/ml. The particle size and zeta potential determinations were performed by using a Malvern particle size analyzer (Zetasizer Nano-ZS, Malvern, UK).

Drug loading capacity and encapsulation efficiency

BSA-NPs (50 mg) were incubated with RhB (5~20 mg) for 2 h. After washing with ultrapure water, the supernatants were collected and analyzed for residual drug concentration by UV-vis analysis.

The drug loading capacity and encapsulation efficiency were calculated as follows:

Encapsulation efficiency (w / w%) = amount of RhB in BSA-NPs/RhB initially added × 100

In vitro drug release behavior

The assay was evaluated in a standard static diffusion cell at a speed of 100 rpm in a shaker at 37°C. The amount of RhB was evaluated using UV-vis spectrometer (560 nm). The amount of RhB released was evaluated at a series of time points, and the release curve was made accordingly.

Cell biocompatibility assay

Cells were seeded in 96-well plates at a density of 1,000 cells/well. BSA-NPs with GA fixation (NP-GA) or heat denaturation (NP-H) were added to each well for a 24-h incubation. Cell viability was determined by CCK-8 assay. Untreated cells served as the control. The morphology of L929 cells in each group was also observed by using a phase contrast microscope.

In vivo assay

Guinea pigs were killed to sample the acoustic bullae (including the RWM). The acoustic bullae were placed in the solution of BSA-NPs and shaking for 30 min at 37°C. The air-dried specimens were observed by SEM.

The penetration of RhB released from the RhB-BSA-NPs was evaluated by live images and microscopes. Guinea pigs were anaesthetized and the RWMs were exposed. The heat-denatured RhB-BSA-NPs and RhB dispersed in PBS were injected slowly into the bullae of

Statistical analysis

The statistical data was presented as the mean value and standard deviation. The analysis of t test was used in SPSS 12.0 to determine significant differences between groups, and P values less than 0.05 were considered statistically significant.

Results and discussion

Morphology of BSA-NPs BSA-NPs were prepared by the desolvation method in high yield (about 95%). Figure 1a and its inset display that BSA-NPs with heat denaturation occur as solid spheres with smooth surface. The size of these spheres determined by dynamic light scattering (DLS) varied from 255 to 825 nm (Figure 1b). The mean value was 492 nm and was larger than the size of 238 nm measured by SEM (analyzed by ImageJ 1.44 software) due to the shrinkage of the particles during dehydration. The difference between SEM and DLS is consistent with the previous literatures [8,15].

As shown in Figure 1c, BSA-NPs with GA fixation were also sphere-shaped with a mean diameter of 320 nm. Therefore, we can conclude that the morphology of BSA-NPs shows no obvious difference in shape even if treated by either heat or GA. However, there was little difference between the particles viewed by the naked eye -the colors of precipitates were yellow (Figure 1d, left) and milk white (Figure 1d, right), respectively.

Drug loading and release study

Rhodamine B was used as a model drug for observation and evaluation of drug loading capacity. The morphology and structure of RhB-loaded NP-H (Figure 2a) did not change in comparison with those of BSA-NPs (Figure 1a). The mean diameter of RhB-loaded NP-H was 636 nm, larger than that of BSA-NPs.  The BSA-NPs and RhB-BSA-NPs had zeta potential values of −15.4 and +4.98 mV, respectively. The potential difference demonstrated that the positively charged RhB had an interaction with the negatively charged BSA [8], which also promoted the attachment of RhB to the BSA. The fluorescent image of the RhB-BSA-NPs ( Figure 2b) further confirmed that RhB had attached to the BSA-NPs. Thus, the model drug and small molecules could affect certain parameters including size and charge of polymers, which was in agreement with the previous reports [16][17][18][19].

The drug loading capacity and encapsulation efficiency of BSA-NPs were also evaluated. The drug loading capacity of BSA was 15.4% for RhB (Figure 2c). The maximum encapsulation efficiency was 40.9% (Figure 2c). It was likely attributed to the electrostatic interaction and hydrophobic interactions between RhB and BSA followed by diffusion of the model drug into the BSA matrix [8,16]. Nevertheless, the drug cannot diffuse into the matrix more after achieving the kinetic equilibrium state. The results in this report were consistent with the report described by Shi and Goh [8].

The in vitro drug release profile of RhB from BSA-NPs is shown in Figure 2d. A good sustained release profile is achieved. The cumulative release of RhB over a period of 150 hours was 429.14 μg, indicating a good affinity between the BSA and RhB. This was governed by Fickian diffusion due to the electrostatic interaction, which restricted the release of positively charged RhB from negatively charged BSA in vitro.

In vitro cytocompatibility study

In vitro experiment of BSA-NPs cross-linked with GA or denatured by heat against L929 cell lines were performed by CCK-8 to evaluate the cytocompatibility. As shown in Figure 3, cell viability of NP-GA was significantly lower (P = 0.001) than that of the control possibly because the water wash in this study was only once. These results indicated that the NP-H had a better cytocompatibility than the NP-GA. The slight cytotoxicity of NP-GA was in agreement with that reported by Speer [20]. There was no statistical difference between the NP-H (P = 0.114) and the control.

The shape of L929 cells incubated with NP-H maintained high viability after the assay (Figure 4d) while round-shaped cells could be observed in the control and NP-GA groups (Figure 4b,c). This indicated that the addition of nontoxic NP-H might provide nutrition and promote cell proliferation due to the hydrophobic domain of such natural protein, just as the silk fibroin particles did [8]. But the nutrition property of BSA on cell proliferation cannot compensate the side effect of GA in  the system, which explained the fact that most cells died with the addition of NP-GA (Figure 4c). The above findings disclosed that BSA was not only a soft material with good biocompatibility but also a nutrition provider. Further studies will focus on the assessment of BSA-NP drug delivery in the treatment of inner ear disorders.

In vivo distribution and drug delivery of BSA-NPs

As for the good cytocompatibility, BSA-NPs with heat denaturation were loaded with RhB and used to evaluate the local drug delivery. Acoustic bullae of guinea pigs with entire RWM were isolated and injected with RhB-BSA-NP (right ear) and RhB solution (left ear). The live images were taken immediately (Figure 5a). Three days later, there was still obvious fluorescent signals with a larger area in the right ear (Figure 5b), which indicated that RhB-BSA-NPs was retained nearby the RWM and RhB possibly diffused into the Eustachian tube and the inner ear. We assumed that the BSA-NPs maybe useful for local drug delivery and controlled release. In the left ear, there were only minimal fluorescent signals of RhB (Figure 5b) because the RhB could penetrate through the RWM by simple diffusion along its concentration gradient.

The guinea pigs were then killed and the temporal bones and RWMs were separated. The nanoparticles still attached on the RWM (Figure 6a). The SEM image revealed that particles aggregated on the osseous spiral lamina and some particles even had penetrated into the cochlea through the RWM (Figure 6b). As previously described that PLGA nanoparticles or lipid core nanocapsules could pass through the RWM and be deposited in various sites of the cochlea [5,[21][22][23], we assumed that the tiny BSA-NPs loaded with RhB could successfully reach the inner ear through the RWM.

Conclusions

In summary, BSA-NPs were fabricated via a desolvation method. The heat-denatured BSA-NPs had a great potential application for local drug delivery into the cochlea to treat inner ear diseases due to the tiny size, good biocompatibility, drug loading capacity, and controlled release profile. Further studies will focus on the evaluation of drug-loaded BSA-NPs, including prednisolone. We will evaluate their pharmacokinetics, pharmacodynamics, and delivery mechanism in animal model. The BSA-NPs also shed light in the treatment of human inner ear diseases.

Figure 1
1Morphology of BSA-NPs with heat denaturation and GA fixation. SEM/TEM images of BSA-NPs with heat denaturation (a) and GA fixation (c) are shown. The size distribution of NP-H evaluated by DLS is shown in (b). The difference between the two kinds of NPs is shown in (d).

Figure 2
2Characteristics of RhB-loaded BSA-NPs. SEM (a), TEM (inset of (a)), and CLSM (b) images of RhB-loaded BSA-NPs denatured by heat are demonstrated. The drug loading capacity, encapsulation efficiency (c), and controlled release profile (d) are shown respectively. the right and left ear, respectively. The left ear injected with RhB solution was the control. In vivo imaging system (Caliper IVIS imaging system, PerkinElmer, Waltham, MA, USA) was used to trace the particles at time points of 0 and 72 h. The RWM was then imaged by fluorescence microscopy and SEM to observe the distribution of RhB and BSA-NPs.

Figure 3
3Cytotoxity evaluation of BSA-NPs fixed by GA or denatured by heat against L929 cells. Each value represents mean ± SD (n = 3) (**P < 0.01).

Figure 4
4Morphology of L929 cells cultured with different conditions. L929 cells cultured in DMEM-10% FBS as the control (a), after performing CCK-8 assay (b), with the addition of NP-GA (c) and NP-H (d), are demonstrated respectively. All images have an original magnification of × 200.

Figure 5
5Live images revealed the distribution of RhB-BSA-NPs. RhB-BSA-NPs with heat denaturation were injected into the right ear, and the images were taken immediately (a) and 72 h later (b). RhB solution injected into the left ear was the control.

Figure 6
6Images of RhB-BSA-NPs adhering on the RWM and osseous spiral lamina. The fluorescent image of RhB-BSA-NPs (a) adhering on the RWM was taken immediately after the surgery. The SEM image of RhB-BSA-NPs (b) deposited on the osseous spiral lamina was taken 3 days later. The aggregated BSA-NPs are shown in the inset (inset of (b)).

Abbreviations BSA: bovine serum albumin; BSA-NP: bovine serum albumin nanoparticles; RWM: round window membrane; PLGA: polylactic/glycolic acid; RhB: rhodamine B; RhB-BSA-NP: RhB-loaded BSA-NP; GA: glutaraldehyde; CCK-8: cell counting kit-8; NP-GA: BSA-NPs with GA fixation; NP-H: BSA-NPs with heat denaturation; DLS: dynamic light scattering; TEM: transmission electron microscope; SEM: scanning electron microscopy.
http://www.nanoscalereslett.com/content/9/1/343
AcknowledgementsWe are grateful for the financial support of the Project in the Eleventh Five-Year Plan of the Second Artillery General Hospital of Chinese People's Liberation Army.Competing interestsThe authors declare that they have no competing interests.Authors' contributions ZY, ZZ, GH, QX, and MY performed the experiments and analyzed the results. ZY and MY conceived and designed the experiments, analyzed the results, and participated in writing the manuscript. All authors read and approved the final manuscript.Submit your manuscript to a journal and benefi t from:7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com
Ablation therapy for the relief of Meniere's disease. H F Schuknecht, Laryngoscope. 66Schuknecht HF: Ablation therapy for the relief of Meniere's disease. Laryngoscope 1956, 66:859-870.

Magnetic nanoparticles: inner ear targeted molecule delivery and middle ear implant. R D Kopke, R A Wassel, F Mondalek, B Grady, K J Chen, J Z Liu, D Gibson, K J Dormer, Audiol Neurotol. 11Kopke RD, Wassel RA, Mondalek F, Grady B, Chen KJ, Liu JZ, Gibson D, Dormer KJ: Magnetic nanoparticles: inner ear targeted molecule delivery and middle ear implant. Audiol Neurotol 2006, 11:123-133.

Kinetics of gentamicin uptake in the inner ear of Chinchilla laniger after middle-ear administration in a sustained-release vehicle. B J Balough, M E Hoffer, D Wester, M J O'leary, C R Brooker, M Goto, Otolaryngol Head Neck Surg. 119Balough BJ, Hoffer ME, Wester D, O'Leary MJ, Brooker CR, Goto M: Kinetics of gentamicin uptake in the inner ear of Chinchilla laniger after middle-ear administration in a sustained-release vehicle. Otolaryngol Head Neck Surg 1998, 119:427-431.

Sustained delivery of lidocaine into the cochlea using polylactic/glycolic acid microparticles. R T Horie, T Sakamoto, T Nakagawa, Y Tabata, N Okamura, N Tomiyama, M Tachibana, J Ito, Laryngoscope. 120Horie RT, Sakamoto T, Nakagawa T, Tabata Y, Okamura N, Tomiyama N, Tachibana M, Ito J: Sustained delivery of lidocaine into the cochlea using polylactic/glycolic acid microparticles. Laryngoscope 2010, 120:377-383.

Distribution of PLGA nanoparticles in chinchilla cochleae. X X Ge, R L Jackson, J Z Liu, E A Harper, M E Hoffer, R A Wassel, K J Dormer, R D Kopke, B J Balough, Otolaryngol. Head Neck Surg. 137Ge XX, Jackson RL, Liu JZ, Harper EA, Hoffer ME, Wassel RA, Dormer KJ, Kopke RD, Balough BJ: Distribution of PLGA nanoparticles in chinchilla cochleae. Otolaryngol. Head Neck Surg 2007, 137:619-623.

Nanoporous peptide particles for encapsulating and releasing neurotrophic factors in an animal model of neurodegeneration. J Tan, Y J Wang, X P Yip, F Glynn, R K Shepherd, F Caruso, Adv Mater. 24Tan J, Wang YJ, Yip XP, Glynn F, Shepherd RK, Caruso F: Nanoporous peptide particles for encapsulating and releasing neurotrophic factors in an animal model of neurodegeneration. Adv Mater 2012, 24:3362-3366.

Protein nanoparticle: a unique system as drug delivery vehicles. M Jahanshahi, Z Babaei, Afr J Biotechnol. 7Jahanshahi M, Babaei Z: Protein nanoparticle: a unique system as drug delivery vehicles. Afr J Biotechnol 2008, 7:4926-4934.

Release and cellular acceptance of multiple drugs loaded silk fibroin particles. P J Shi, Jch Goh, Int J Pharm. 420Shi PJ, Goh JCH: Release and cellular acceptance of multiple drugs loaded silk fibroin particles. Int J Pharm 2011, 420:282-289.

Desolvation process and surface characterisation of protein nanoparticles. C Weber, C Coester, J Kreuter, K Langer, Int J Pharm. 194Weber C, Coester C, Kreuter J, Langer K: Desolvation process and surface characterisation of protein nanoparticles. Int J Pharm 2000, 194:91-102.

Make conjugation simple: a facile approach to integrated nanostructures. Y Xu, S Palchoudhury, Y Qin, T Macher, Y Bao, Langmuir. 28Xu Y, Palchoudhury S, Qin Y, Macher T, Bao Y: Make conjugation simple: a facile approach to integrated nanostructures. Langmuir 2012, 28:8767-8772.

Synthesis of protein-based, rod-shaped particles from spherical templates using layer-by-layer assembly. Z M Zhou, A C Anselmo, S Mitragotri, Adv Mater. 25Zhou ZM, Anselmo AC, Mitragotri S: Synthesis of protein-based, rod-shaped particles from spherical templates using layer-by-layer assembly. Adv Mater 2013, 25:2723-2727.

Bovine serum albumin nanoparticle vaccine reduces lung pathology induced by live Pseudomonas aeruginosa infection in mice. N F Rodrigues, E T Bernardes, R P Rocha, Vaccine. 31Rodrigues NF, Bernardes ET, Rocha RP: Bovine serum albumin nanoparticle vaccine reduces lung pathology induced by live Pseudomonas aeruginosa infection in mice. Vaccine 2013, 31:5062-5066.

Albumin-based nanoparticles as potential controlled release drug delivery systems. A O Elzoghby, W M Samy, N A Elgindy, J Control Release. 157Elzoghby AO, Samy WM, Elgindy NA: Albumin-based nanoparticles as potential controlled release drug delivery systems. J Control Release 2012, 157:168-182.

Impact of albumin on drug delivery -new applications on the horizon. B Elsadek, F Kratz, J Control Release. 157Elsadek B, Kratz F: Impact of albumin on drug delivery -new applications on the horizon. J Control Release 2012, 157:4-28.

Pullulan acetate nanoparticles prepared by solvent diffusion method for epirubicin chemotherapy. H Z Zhang, F P Gao, L R Liu, X M Li, Z M Zhou, X D Yang, Q Q Zhang, Colloids Surf B: Biointerfaces. 71Zhang HZ, Gao FP, Liu LR, Li XM, Zhou ZM, Yang XD, Zhang QQ: Pullulan acetate nanoparticles prepared by solvent diffusion method for epirubicin chemotherapy. Colloids Surf B: Biointerfaces 2009, 71:19-26.

Controlling silk fibroin particle features for drug delivery. A S Lammel, X Hu, S H Park, D L Kaplan, T R Scheibel, Biomaterials. 31Lammel AS, Hu X, Park SH, Kaplan DL, Scheibel TR: Controlling silk fibroin particle features for drug delivery. Biomaterials 2010, 31:4583-4591.

High-yield fabrication of PLGA non-spherical microarchitectures by emulsion-solvent evaporation method. R F Li, X M Li, L R Liu, Z M Zhou, H B Tang, Q Q Zhang, Macromol Rapid Commun. 31Li RF, Li XM, Liu LR, Zhou ZM, Tang HB, Zhang QQ: High-yield fabrication of PLGA non-spherical microarchitectures by emulsion-solvent evaporation method. Macromol Rapid Commun 2010, 31:1981-1986.

Formation of poly(L, D-lactide) spheres with controlled size by direct dialysis. M Liu, Z M Zhou, X F Wang, J Xu, K Yang, Q Cui, X Chen, M Y Cao, J Weng, Q Q Zhang, Polymer. 48Liu M, Zhou ZM, Wang XF, Xu J, Yang K, Cui Q, Chen X, Cao MY, Weng J, Zhang QQ: Formation of poly(L, D-lactide) spheres with controlled size by direct dialysis. Polymer 2007, 48:5767-5779.

Non-spherical racemic polylactide microarchitectures formation via solvent evaporation method. Z M Zhou, J Xu, X Q Liu, X M Li, S Y Li, Yang K Wang, X F Liu, M Zhang, Q Q , Polymer. 50Zhou ZM, Xu J, Liu XQ, Li XM, Li SY, Yang K, Wang XF, Liu M, Zhang QQ: Non-spherical racemic polylactide microarchitectures formation via solvent evaporation method. Polymer 2009, 50:3841-3850.

Biological effects of residual glutaraldehyde in glutaraldehyde-tanned collagen biomaterials. D P Speer, M Chvapil, C D Eskelson, J Ulreich, J Biomed Mater Res. 14Speer DP, Chvapil M, Eskelson CD, Ulreich J: Biological effects of residual glutaraldehyde in glutaraldehyde-tanned collagen biomaterials. J Biomed Mater Res 1980, 14:753-764.

Drug delivery to the cochlea using PLGA nanoparticles. T Tamura, T Kita, T Nakagawa, T Endo, T S Kim, T Ishihara, Y Mizushima, M Higaki, J Ito, Laryngoscope. 115Tamura T, Kita T, Nakagawa T, Endo T, Kim TS, Ishihara T, Mizushima Y, Higaki M, Ito J: Drug delivery to the cochlea using PLGA nanoparticles. Laryngoscope 2005, 115:2000-2005.

Inner ear biocompatibility of lipid nanocapsules after round window membrane application. Y Zhang, W K Zhang, M Löbler, K P Schmitz, P Saulnier, T Perrier, I Pyykkö, J Zou, Int J Pharm. 404Zhang Y, Zhang WK, Löbler M, Schmitz KP, Saulnier P, Perrier T, Pyykkö I, Zou J: Inner ear biocompatibility of lipid nanocapsules after round window membrane application. Int J Pharm 2011, 404:211-219.

Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation. J Zou, P Saulnier, T Perrier, Y Zhang, T Manninen, E Toppila, I Pyykkö, 10.1186/1556-276X-9-343J Biomed Mater Res Part B Appl Biomater. 87Zou J, Saulnier P, Perrier T, Zhang Y, Manninen T, Toppila E, Pyykkö I: Distribution of lipid nanocapsules in different cochlear cell populations after round window membrane permeation. J Biomed Mater Res Part B Appl Biomater 2008, 87B:10-18. doi:10.1186/1556-276X-9-343

Bovine serum albumin nanoparticles as controlled release carrier for local drug delivery to the inner ear. Yu , Nanoscale Research Letters. 9343Cite this article asCite this article as: Yu et al.: Bovine serum albumin nanoparticles as controlled release carrier for local drug delivery to the inner ear. Nanoscale Research Letters 2014 9:343.